JP4338402B2 - N4 virus single-stranded DNA-dependent RNA polymerase - Google Patents

Description

(Background of the Invention)
This application claims priority to US Provisional Patent Application No. 60 / 292,845, filed May 22, 2001, the entire disclosure of which is hereby incorporated by reference in its entirety. Grant number R01 A1 12575 from National Institute of Health allows the government to have rights in this invention.

(I. Field of the Invention)
The present invention relates generally to RNA polymerases. More particularly, the present invention provides a bacteriophage N4 viral RNA polymerase for synthesizing RNA of a desired sequence using a single stranded DNA template.

(II. Explanation of related technology)
Expression of a gene encoding a protein in a host cell involves transcription of messenger RNA (mRNA) from DNA by an RNA polymerase enzyme. Subsequently, the mRNA is processed. This processing involves the recognition of the 3′UTR region by polyadenylation enzymes and the addition of a tail of a polyadenylated nucleotide to the 3 ′ end of the mRNA. After transcription, the mRNA encounters a ribosome that binds to the 5′UTR region of the mRNA and translocates along the mRNA in the 3 ′ direction. During transfer, amino acids are sequentially added to each other to form a polypeptide product of the gene encoding the protein. For prokaryotic transcription-translation, the Shine-Dalgarno sequence of bacterial mRNA located 6-9 nucleotides before the translation start site can be used for ribosome loading. This sequence is complementary to the sequence on the 3 'end of 16S rRNA and stimulates the ribosome to bind to mRNA. Base pairing between the Shine-Dalgarno sequence and the RNA sequence serves to align the starting AUG for decoding.

  Transcription of DNA into mRNA is regulated by the promoter region of DNA. This promoter region contains a base sequence that indicates that RNA polymerase binds to DNA and initiates transcription of mRNA using one of the DNA strands as a template to create the corresponding complementary strand of RNA. . RNA polymerases from different species typically recognize promoter regions consisting of different sequences. In order for a protein-encoding gene to be expressed in a host cell, the promoter driving the transcription of the protein-encoding gene must be recognized by the host RNA polymerase or the gene encoding the protein can be translated. An RNA polymerase that recognizes the driving promoter must be provided to the host cell (US Pat. No. 6,218,145).

  Most DNA-dependent RNA polymerases read double-stranded DNA and limit RNA synthesis to systems where double-stranded DNA templates are available. The synthesis of RNA using single-stranded DNA is not common. Synthesis of RNA using a single stranded DNA template immobilized on a solid support is described in US Pat. No. 5,700,667.

  Accordingly, the present invention provides an RNA polymerase that reads single-stranded DNA. RNA polymerases are also provided in which a promoter sequence is present upstream of the translation initiation site and is therefore not transcribed by the polymerase.

(Summary of the Invention)
The present invention provides novel N4 viral RNA polymerase (vRNAP) and mini-vRNA polymerase and methods of use thereof. The novel polymerase is described by an isolated nucleic acid comprising a region encoding a polypeptide having an amino sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 15. The nucleic acid can comprise the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 14. vRNAP polymerase and mini-vRNA polymerase transcribe nucleic acid operably linked to an N4 promoter (eg, the P2 promoter of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29). The promoter of SEQ ID NO: 16 or SEQ ID NO: 28 is preferred.

  One aspect of the invention includes a recombinant host cell comprising a DNA segment encoding N4 viral RNA polymerase. The DNA segment is either single-stranded or double-stranded, and the polypeptide encoded by this DNA segment is preferably SEQ ID NO: 4 or SEQ ID NO: 6. The recombinant host cell is E. coli. E. coli cells. Another aspect of the invention includes a recombinant vector comprising a DNA segment encoding an N4 viral RNA polymerase polypeptide under the control of a promoter.

  Yet another aspect of the invention includes an isolated polynucleotide comprising a sequence that is identical to or complementary to at least 14 consecutive nucleotides of SEQ ID NO: 1. The polynucleotide comprises at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 75, at least 100, at least 150, at least 200, at least 250, at least of SEQ ID NO: 1. It may comprise 300, at least 400, at least 600, at least 800, at least 1000, at least 2000, at least 3000, at least 3300 or more consecutive nucleotides. The polynucleotide can comprise all contiguous nucleotides of SEQ ID NO: 3 or all nucleotides of SEQ ID NO: 1.

  Similarly, the polynucleotide comprises at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 75, at least 100, at least 150, at least 200, at least of SEQ ID NO: 1. 250, at least 300, at least 400, at least 600, at least 800, at least 1000, at least 2000, at least 3000, at least 3300 or more complementary to at least 20, at least 25, at least 30, at least 35, at least 40 , At least 45, at least 50, at least 60, at least 75, at least 100, at least 150, at least 200, less Also 250, at least 300, at least 400, at least 600, at least 800, at least 1000, at least 2000, at least 3000, preferably contains at least 3300 or more contiguous nucleotides.

  Another aspect of the invention includes a purified N4 viral RNA polymerase comprising at least 20 consecutive amino acids of SEQ ID NO: 2. The polymerase is at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 of SEQ ID NO: 2. , At least 600, at least 800, at least 1000 or more consecutive amino acids.

  Yet another aspect of the invention includes an isolated nucleic acid comprising a region encoding a polypeptide comprising at least 6 consecutive amino acids of SEQ ID NO: 2, wherein the polypeptide is suitable under suitable reaction conditions. And has RNA polymerase activity. The polypeptide is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 15. Preferably, it comprises 45, at least 50, at least 60, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 600, at least 800, at least 1000 or more consecutive amino acids. The encoded polypeptide can have at least one hexahistidine tag or other tag. The polypeptide can be a variant of the peptide found in SEQ ID NO: 2 or SEQ ID NO: 4 (eg, an enzyme containing an amino acid substitution at position Y678).

  One embodiment of the invention includes a method of making RNA. The method includes: (a) obtaining an N4 viral RNA polymerase (ie, polypeptide); (b) obtaining a DNA, wherein the DNA preferably comprises an N4 viral RNA polymerase promoter sequence. (C) mixing the RNA polymerase with the DNA; and (d) culturing the DNA with the RNA polymerase under conditions effective to allow RNA synthesis. . Optionally, the method can include synthesizing a polynucleotide comprising a modified ribonucleotide or modified deoxynucleotide. This DNA is preferably single-stranded DNA or denatured double-stranded DNA. Step (c) can be done in a host cell (eg, an E. coli host cell).

  The amino acid sequence of the RNA polymerase is preferably the sequence essentially shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 15, or the polymerase of SEQ ID NO: 4 or SEQ ID NO: 6 It is a mutant form. This mutation can be, for example, at position number Y678. RNA transcription can include derivatized nucleotides.

  One aspect of the invention involves using an N4 vRNAP promoter to direct transcription. This promoter is preferably the N4 promoter shown in SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29. The P2 promoter of SEQ ID NO: 16 or SEQ ID NO: 28 is preferred. This promoter sequence can be upstream of the transcription start site. This promoter comprises a central position of a loop of 3-5 bases and / or a stem of 2-7 base pairs in length with a purine next to its center and a set of inverted repeats forming a hairpin with that loop. obtain.

Preferred conditions for the transfer method claimed herein include a pH between 6 and 9 in step (c), with a pH between 7.5 and 8.5 being more preferred. Mg ⁺² or Mn ⁺² (preferably Mg ⁺² ) can be mixed. Preferred temperatures for this reaction are 25 ° C to 50 ° C, more preferably in the range of 30 ° C to 45 ° C, and most preferably in the range of 32 ° C to 42 ° C. This mixing can be done in vivo or in vitro.

  One aspect of the invention also includes translation of RNA after transcription. A receptor gene (eg, an α-peptide of β-galactosidase) can be used. Transcription is performed in E.C. Preferably, the method comprises a step of mixing the polymerase and DNA with a single-stranded binding protein of E. coli (EcoSSB), an SSB protein homologous to EcoSSB, or a naturally occurring SSB protein or chimeric SSB protein homologous to EcoSSB.

  The DNA mixed with the RNA polymerase of the present invention can be single-stranded linear DNA or can be single-stranded circular DNA (eg, bacteriophage M13 DNA). The DNA can be a denatured DNA (eg, single stranded denatured DNA, double stranded linear denatured DNA or double stranded circular denatured DNA. The DNA can also be double stranded under certain conditions. The RNA can be pure RNA or can contain modified nucleotides, and the mixed RNA-DNA oligonucleotide can also use the Y678F mutant mini-vRNAP of the present invention (SEQ ID NO: 8). Can be synthesized.

  Yet another aspect of the invention is a transcription method in which EcoSSB is not mixed with RNA polymerase and DNA; the product of this method is a DNA / RNA hybrid.

The synthesized RNA can include a detectable label (eg, a fluorescent tag, biotin, digoxigenin, 2 ′ fluoronucleoside triphosphate, or a radiolabel (eg, ³⁵ S-label or ³² P-label)). The synthesized RNA can be adapted for use as a probe for blotting experiments or in situ hybridization. Nucleoside triphosphates (NTPs) or derivatized NTPs can be incorporated into RNA and optionally have a detectable label. Deoxynucleoside triphosphates can be incorporated into RNA.

  This RNA can be adapted for use for NMR structure determination. Short RNA (eg, RNA between 10 and 1000 bases or between 10 and 300 bases) can be used. The RNA can be adapted for use in spliceosome assembly, splicing reactions, or antisense experiments. The RNA can also be adapted for use in probing complementary nucleotide sequences or as a probe in RNase protection studies.

  Yet another aspect of the invention involves delivering RNA to a cell after transcription of the RNA. This delivery can be microinjection. Another aspect of the invention involves amplifying RNA after transcription.

  Another embodiment of the invention includes a method of making RNA, the method comprising: (a) obtaining an N4 viral RNA polymerase; (b) obtaining a single stranded DNA oligonucleotide comprising: Wherein the oligonucleotide comprises an N4 viral RNA polymerase promoter sequence; (c) mixing the RNA polymerase with the oligonucleotide; and (d) conditions effective to allow RNA synthesis. And culturing the RNA polymerase and the oligonucleotide. This polymerase preferably has the amino sequence shown in SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. In this embodiment, it is preferred that the DNA has between 20 and 200 bases.

  Yet another embodiment of the invention includes a method of making RNA, the method comprising: (a) obtaining N4 viral RNA polymerase; (b) obtaining single stranded DNA comprising: Wherein the DNA comprises an N4 viral RNA polymerase promoter sequence; (c) obtaining a ribonucleoside triphosphate (XTP) or derivatized ribonucleoside triphosphate derivative; (d) the RNA polymerase, the DNA, and Mixing XTP; and (e) culturing the RNA polymerase and the oligonucleotide under conditions effective to allow RNA synthesis, wherein the RNA is derivatized RNA A step. This RNA polymerase is preferably the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6, or a variant of the polymerase of SEQ ID NO: 4 or SEQ ID NO: 6 (eg, a variant having a mutation at position No. Y678) or SEQ ID NO: 8 Of polymerase.

  Another embodiment of the invention includes a method for protein synthesis in vivo or in vitro, which comprises: (a) the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, or a variant thereof. (B) obtaining DNA, wherein the DNA comprises an N4 viral RNA polymerase promoter sequence; (c) mixing the RNA polymerase with the DNA; d) culturing the RNA polymerase and the DNA under conditions effective to allow RNA synthesis; and (e) in vivo or in vitro under conditions effective to allow protein synthesis. And culturing the RNA. Step (e) includes using a 1 plasmid system or a 2 plasmid system, wherein the reporter gene and the RNA polymerase gene are located on the same plasmid.

  Yet another embodiment of the invention includes a method of making an N4 mini vRNAP, the method comprising: (a) expressing vRNAP, wherein vRNAP is SEQ ID NO: 2, SEQ ID NO: 4 , Having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 15 or a variant thereof; and (b) purifying vRNAP. Expression of vRNAP can occur in bacteria, yeast, CHO host cells, Cos host cells, HeLa host cells, NIH3T3 host cells, Jurkat host cells, 293 host cells, Saos host cells, or PC12 host cells. A promoter such as pBAD can be used to make vRNAP in bacterial cells. Any other promoter suitable for the host cell line used can be used when expressing vRNAP in other host cells. The polymerase may have a specific recombination sequence that can be used for purification of the polymerase. The vRNAP can have at least one of hexahistidine, FLAG, hemagglutinin or c-myc tag, or no tag.

(Description of Exemplary Embodiments)
The present invention overcomes the shortcomings in the art by providing a stable RNA polymerase that uses single-stranded DNA and provides a unique system for synthesizing RNA of the desired sequence. RNA polymerases and mini-vRNA polymerases are used for the synthesis of desired RNA: DNA hybrids for use as probes in DNA or RNA RNase protection studies, in situ hybridization studies, and Southern and Northern blot analysis. It can be used for structure determination, for in vitro studies of spliceosome assembly, splicing reactions and antisense experiments, for in vitro translation or microinjection, and for RNA synthesis for nucleic acid amplification. The present invention allows the synthesis of derivatized RNA and can use ssDNA in the form of single stranded p-ligonucleotides to either modify the DNA or clone the DNA into an M13 template.

(I. RNA polymerase)
(A. Structure and promoter recognition of DNA-dependent RNA polymerase)
Examination of DNA-dependent RNA polymerases of phage, archaea, eubacteria, eukaryotes and viruses revealed the existence of two enzyme families. This eubacteria, eukaryote, archaea, chloroplast and vaccinia virus RNA polymerase is a complex multi-subunit enzyme (5-14 subunits) composed of two large subunits, one of which is , Some subunits of medium molecular weight (30-50 kDa) and others are subunits of low molecular weight (<30 kDa) (Archambault et al., 1993; Record et al., 1995). Eubacterial RNA polymerase is the simplest with an α2ββ ′ core structure. Sequence comparison of the genes encoding the different subunits of these enzymes revealed the following: 1) in 8 segments (AH) between β ′ and the largest subunit of other RNA polymerases Sequence homology; 2) sequence homology occurring in 9 segments (AI) between β and the next largest subunit of other RNA polymerases; 3) α and RNA polymerases I, II and III Sequence homology in three segments (1.1, 1.2 and 2) between subunits in (Puhler et al., 1989; Sweeters et al., 1987). As a matter of course, yeast RNAP II and E.I. The crystal structure of the E. coli RNAP core revealed significant similarities (Zhang et al., 1999; Cramer et al., 2001).

  In contrast, members of the phage T7-like (T3, SP6) family of RNA polymerases consist of a single (approximately 100 kDa) polypeptide that catalyzes all functions required for accurate transcription ( Cheetham, et al., 2000). Heterodimeric bacteriophage N4 RNAP II, nuclear-encoded mitochondrial RNA polymerase, and Arabidopsis chloroplast RNA polymerase show sequence similarity to phage RNA polymerase (Cermakian et al., 1996; Hedtke et al., 1997; Zehring et al., 1983). These sequence motifs A and C contain the two aspartic acids required for the catalyst, and motif B is conserved in a polymerase that uses DNA as a template (Delarue et al., 1990). The crystal structure of T7 RNAP is similar to a “cupped right hand” with “palm”, “finger” and “thum” subdomains (Sousa et al., 1993). . Two catalytic aspartames are present in the “palm” of the structure. This structure is shown in E.I. shared by the polymerase domains of E. coli DNA polymerase I and HIV reverse transcriptase (Sousa, 1996). Genetic, biochemical and structural information indicates that T7 RNA polymerase contains additional structures that provide early RNA binding, promoter recognition, dsDNA unwinding, and RNA: DNA hybrid unwinding (Cheetham et al. 2000; Sousa, 1996).

Class I and class II RNA polymerases recognize specific sequences on type B double stranded DNA, called promoters. (Excluding those that are recognized by the sigma ⁵⁴⁾ eubacteria promoter is characterized by two regions of the following sequence homology: -10 and -35 hexamers (Gross et al., 1998). The specificity of promoter recognition is provided to the core enzyme by the σ subunit, which specifically interacts with the -10 and -35 sequences via two different DNA binding domains (Gross et al., 1998). This modular promoter structure is also present with promoters for eukaryotic RNA polymerases I, II and III. Transcription factors TFIIIA and TFIIIC directly recognize RNAP III against two distinct sequences (boxes A and C separated by a defined interval) at the 5S gene promoter, while transcription factors TFIIIB and TFIIIC are tRNA promoters. Direct recognition of this enzyme for blocks A and B, separated by a variable distance (31-74 bp) (Paule et al., 2000). Sequences important for RNAP I transcription initiation at the human rRNA promoter are also restricted to the following two regions: a “core” region located at −40 to +1, and an “upstream” region present at −160 to −107. Region (Paule et al., 2000). Assembly of the initiation complex with the RNAP II promoter requires several common transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIIH). Recognition includes the following three core elements: a TATA box located at -30 and recognized by TBP, a start element located near -1, and a downstream promoter element near +30 (Roeder, 1996).

  The promoters for T7-like RNAPase and mitochondrial RNAPase are simpler. The T7 type RNAP promoter spaced a continuous highly conserved 23 bp region extending from positions -17 to +6 compared to the start site of transcription (+1) (Rong et al., 1998). The yeast mitochondrial RNAP promoter is still smaller and extends from -8 to +1 (Shadel et al., 1993). One exception is the promoter for N4 RNAP II, which is limited to two blocks of the following conserved sequences: a / tTTTA at +1 and AAGACCTG (Abravaya et al.) Present 18-26 bp upstream of +1. 1990).

The activity of the multi-subunit class of RNA polymerase is enhanced by an activator with a weak promoter. Transcriptional activators generally bind to specific sites upstream of double-stranded DNA in the -35 region (excluding the T4 sliding clamp activator) or in the case of enhancers over long distances (Sanders et al., 1997) . Activators regulate transcription by increasing binding (formation of closed complexes), or the isomerization process of transcription through interactions with the α or σ subunits of RNAP (formation of open complexes) (Hochschild et al., 1998). One exception is that of E. coli with the bacteriophage N4 late promoter. N4SSB, an activator of E. coli RNAPσ ⁷⁰ , which activates transcription through direct interaction with the β ′ subunit of RNAP in the absence of DNA binding (Miller et al., 1997). .

  Proteins that bind with high affinity to ssDNA but without sequence specificity have been purified and characterized from several prokaryotes, eukaryotes, and their viruses (Chase et al., 1986). These proteins (SSBs) are required for replication, recombination and repair and bind stoichiometrically and (in many cases) cooperatively with ssDNA as a result of replication, repair and recombination. Covers single-stranded regions of transient DNA that normally occur in vivo. Binding to DNA results in the removal of the hairpin structure found on ssDNA, providing a developed higher order structure for proteins involved in DNA metabolism. Several lines of evidence suggest that single-stranded DNA binding proteins play a more bold role in intracellular processes. Genetic and biochemical evidence indicates that these proteins relax to a large number of protein-protein interactions, including transcriptional activation (Rothman-Denes et al., 1999).

(B. Bacteriophage N4 virion RNA polymerase)
Bacteriophage N4 virion RNA polymerase (N4 vRNAP) is present in the N4 virion and is E. coli early in infection. injected into E. coli, where it results in transcription of the N4 early gene (Falco et al., 1977; Falco et al., 1979; Malone et al., 1988). The N4 vRNAP gene maps to the late region of the N4 genome (Zivin et al., 1981). N4 vRNAP purified from virions is composed of a single polypeptide with an apparent molecular weight of approximately 320,000 kD (Falco et al., 1980). In contrast to other DNA-dependent RNAPases, N4 vRNAP recognizes a promoter on a single-stranded template (Falco et al., 1978). These promoters are characterized by conserved sequences and a 5 bp stem structure, a 3 base loop hairpin structure (FIG. 1) (Haynes et al., 1985; Glucksmann et al., 1992). In vivo, E. coli. E. coli gyrase and single-stranded binding proteins are required for transcription by N4 vRNAP (Falco et al., 1980; Markiewicz et al., 1992).

Sequencing of the N4 vRNAP gene revealed an ORF that encodes a 3,500 amino acid long protein (SEQ ID NOs: 1-2). Sequence inspection revealed no extensive homology to either the multi-subunit or the T7-like family of RNA polymerases. However, there are three motifs (FIG. 2A): T / DxxGR motif found in DNA-dependent polymerase, and motif B (R _X3 K _X6-7 YG), Pol I and Pol α DNA polymerase and T7-like One of three motifs common to RNA polymerases.

(C. Transcription using N4 vRNAP)
RNA synthesis requires RNA polymerase, DNA template, activation precursors (ribonucleoside triphosphates ATP, GTP, UTP and CTP (XTP)), and divalent metal ions (eg, Mg ²⁺ or Mn ²⁺ ) . The metal ion Mg ²⁺ is highly preferred. RNA synthesis begins at the promoter site of DNA. This site contains sequences that are recognized and bound by RNA polymerase. RNA synthesis proceeds until the termination site is reached. The N4 vRNAP termination signal contains a hairpin loop that forms in newly synthesized RNA, followed by a single letter of uracil (poly U). Termination signal sequences for vRNAP present in the N4 genome include SEQ ID NOs: 21-26. These N4 vRNAP termination signals possess all the characteristics of eubacterial sequence-dependent terminators.

  Ribonucleoside triphosphates can be derivatized with, for example, biotin. Derivatized XTP can be used for the preparation of derivatized RNA. Exemplary methods for making derivatized XTP are disclosed in detail in Rashtian et al. (1992), incorporated herein by reference.

  Variable length single stranded DNA can be used as a template for RNA synthesis using N4 vRNAP or mini-vRNAP. Medium length oligonucleotides and polynucleotides can be used. One particular single stranded DNA that can be used is M13 DNA. M13 genomic DNA is a double-stranded DNA that is It resides temporarily within E. coli and is packaged in phage particles as small, single-stranded circular DNA. M13 phage particles are secreted by infected cells and single stranded DNA can be purified from these particles for use as a transcription template. Initially the M13 phage vector required knowledge of the action of phage biology and was first used to create single stranded DNA molecules for DNA sequences. An M13-derived cloning vector called “phagemid” utilizes M13 replication to produce single-stranded molecules, but can be propagated as a conventional ColE1-based replicating double-stranded plasmid.

  EcoSSB is essential for N4 vRNAP transcription in vivo (Falco et al., 1978; Glucksmann et al., 1992, incorporated herein by reference). EcoSSB is a specific activator of N4 vRNAP on single-stranded DNA templates and supercoiled double-stranded DNA templates. Unlike other SSBs, EcoSSB does not adopt the N4 vRNAP promoter hairpin structure (Glucksmann-Kuis et al., 1996). EcoSSB has a high specificity for N4 vRNAP and a mini-vRNAP that arises from the EcoSSB brain to stabilize the template strand hairpin, while the non-template strand hairpin is destabilized. Other single-stranded DNA binding proteins destabilize template strand hairpins (Glucksmann-Kuis et al., 1996; Dai et al., 1998). If EcoSSB is not used in N4 vRNAP transcription in vitro, DNA: RNA hybrids are formed, preventing template reuse.

(II. Gene and DNA segment)
Important aspects of the invention include isolated DNA segments, and recombinant vectors encoding N4 vRNAP (more specifically, mini-vRNAP or mini-vRNAP variants), as well as wild type, polymorphism or mutation The present invention relates to the production and use of recombinant host cells through the application of DNA technology to express somatic vRNAP. Another aspect of the invention relates to recombinant vectors encoding isolated nucleic acid segments and vRNAP. The sequences of SEQ ID NOs: 1, 3, 4, 7, 14 and biologically functional equivalents thereof are used in recent inventions. Single stranded DNA oligonucleotides and polynucleotides can be used as DNA templates.

  The present invention relates to an isolated nucleic acid segment capable of expressing a protein, polypeptide or peptide having RNA polymerase activity. As used herein, the term “nucleic acid segment” refers to a nucleic acid molecule isolated from free whole genomic DNA of a particular species. Thus, a nucleic acid segment encoding vRNAP refers to a nucleic acid segment comprising a wild-type, polymorphic or mutant vRNAP coding sequence, still isolated from whole bacterial or N4 phage genomic DNA, or purified separately. The Indeed, the term “nucleic acid segment” refers to nucleic acid segments and smaller fragments of such segments, or recombinant vectors, such as plasmids, cosmids, phages, viruses, and the like.

  Similarly, a nucleic acid segment comprising an isolated or purified vRNAP gene refers to a nucleic acid segment comprising: vRNAP protein, polypeptide coding sequence or peptide coding sequence, and in certain aspects, regulatory sequences, other naturally occurring sequences. A sequence substantially isolated from an existing gene or protein coding sequence. In this regard, the term “gene” is used merely to refer to a functional protein, polypeptide or peptide coding unit. As will be appreciated by those skilled in the art, this functional term is adapted to express or express genomic sequences, cDNA sequences, and variants of proteins, polypeptides, domains, peptides, vRNAP and vRNAP coding sequences. Includes both manipulated segments that can be performed.

  “Substantially isolated from other coding sequences” means that the gene of interest (in this case vRNAP, and more particularly the mini-vRNAP gene) forms an important part of the coding region of the nucleic acid segment, and It means that the nucleic acid segment does not contain the majority of naturally occurring coding DNA (eg, large chromosomal fragments, or other functional genes or cDNA coding regions). Of course, this refers to an originally isolated DNA segment and does not include genes or coding regions later added to the segment by human hands.

  The term “essentially described in SEQ ID NO: 2”, for example, actually corresponds to a part of SEQ ID NO: 2 and is not identical to the amino acid of SEQ ID NO: 2 or its biologically functional By equivalent is meant a sequence having a relatively small number of amino acids. This applies for all peptide and protein sequences herein (eg, the sequences of SEQ ID NOs: 4, 6, 8 and 15).

  The term “biologically functional equivalent” is well understood in the art and is defined in more detail herein. Therefore, about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62 %, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, or about 99%, and any range of derivatives thereof (eg, identical or functionally equivalent to the amino acid of SEQ ID NO: 2 A sequence having from about 70% to about 80%, more preferably from about 81% to about 90%, even more preferably from about 91% to about 99%) Sequence, provided that the biological activity of the protein is maintained. In certain embodiments, the biological activity of the vRNAP protein, polypeptide or peptide, or biologically functional equivalent comprises a transcript. A preferred transcriptional activity that can be carried by a vRNAP protein, polypeptide or peptide, or biologically functional equivalent is RNA synthesis using single-stranded N4 vRNAP promoter-containing DNA as a template.

  In other specific embodiments, the present invention relates to isolated nucleic acid segments and recombinant vectors, which comprise in its sequence essentially the nucleic acid sequence set forth in SEQ ID NO: 1. The term “essentially described in SEQ ID NO: 1” is used at the same intervals as above and actually corresponds to a part of SEQ ID NO: 1 and is not identical to the codon of SEQ ID NO: 1 or functionally. Means a sequence having several non-equivalent codons. Again, nucleic acid segments encoding proteins, polypeptides or peptides that exhibit RNAP activity are most preferred.

  The term “functionally equivalent codon” refers to a codon that encodes the same amino acid (eg, six codons for arginine and serine) and / or refers to a codon that encodes a biologically equivalent amino acid. As used herein. For optimization of vRNAP expression in human cells, codons are shown in Table 1 with preferred use from left to right. Thus, the most preferred codon for arginine is “GCC” and the least preferred is “GCG” (see Table 1 below). Codon usage tables for various organisms and organelles can be found on the website http: // www. kazusa. or. jp / codon /, allowing one skilled in the art to optimize codon usage for expression in various organisms using the disclosure herein. Thus, the codon usage table is based on the preferred codon usage tables known to those skilled in the art, based on other animals, and other organisms (eg, prokaryotes (eg, eubacteria), archaea, eukaryotes (eg, protists) It is contemplated that it can be optimized for organisms, plants, fungi, animals), viruses, etc.), and organelles containing nucleic acids (eg, mitochondria or chloroplasts).

アミノ酸配列および核酸配列が、さらなる残基（たとえば、さらなるＮ末端もしくはＣ末端アミノ酸、または５’もしくは３’配列）を含み得、そして、配列が、生物学的タンパク質活性、ポリペプチドもしくはペプチド活性の維持を含む、上記の基準に適合する限り、なお本明細書中に開示される配列の１つに基本的に記載されることがまた、理解される。末端配列の添加は、特に、例えば、コード領域の５’部分または３’部分のいずれかに隣接する種々の非コード配列を含み得るか、あるいは種々の内部配列（すなわち、遺伝子内に存在することが公知であるイントロン）を含み得る核酸配列に適用する。

Amino acid and nucleic acid sequences can include additional residues (eg, additional N-terminal or C-terminal amino acids, or 5 ′ or 3 ′ sequences), and the sequence can be of biological protein activity, polypeptide or peptide activity. It will also be understood that as long as it meets the above criteria, including maintenance, it will still be described essentially in one of the sequences disclosed herein. The addition of terminal sequences can include, for example, various non-coding sequences adjacent to either the 5 ′ part or 3 ′ part of the coding region, or various internal sequences (ie, present within the gene). Applies to nucleic acid sequences that may contain known introns).

  The degeneracy of the genetic code is possible, excluding intron regions or adjacent regions, and is approximately 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40 %, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73% 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 Sequences having%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99%, and herein Any range of derivatives (eg, about 50% to about 80%, more preferably about 81% to about 90%, even more preferably about 91% to about 99% amino acids) 1 "sequence.

(A. Nucleic acid hybridization)
The nucleic acid sequences disclosed herein have a variety of uses. A contiguous sequence derived from a vRNAP nucleic acid sequence can be used, for example, as a template for synthesizing vRNAP.

  In general, the present invention also encompasses DNA segments that are complementary or essentially complementary to the sequences set forth in SEQ ID NOs: 1, 3, 5, 7, and 14. Nucleic acid sequences that are “complementary” are sequences that can be base-paired according to standard Watson-Crick complementarity rules. As used herein, the term “complementary sequence” can be evaluated by the same nucleotides as compared to the above, or under stringent conditions as described herein under SEQ ID NO: 1. Complementary nucleic acid sequence that can be defined to be capable of hybridizing to a nucleic acid segment of

  As used herein, “DNA / RNA hybrid” is understood to mean that a single strand of RNA hybridizes to a single strand of DNA.

  As described herein, the term “appropriate reaction conditions” means temperature, pH, buffer, and other parameters that are adjusted to optimize reaction rate and yield.

  As used herein, “hybridization”, “hybrid”, or “can hybridize” refers to a double-stranded or triple-stranded molecule, or a partial double-stranded or triple-stranded nature. Is understood to mean the formation of molecules having The terms “hybridization”, “hybridize” or “can hybridize” refer to the terms “stringent requirements” or “high stringency” and the terms “low stringency” or “low stringency conditions”. Is included.

  As used herein, “stringent conditions” or “high stringency” allows for hybridization between or within one or more nucleic acid molecules comprising complementary sequences, but of random sequences. This is a condition for preventing hybridization. If there is any mismatch between the nucleic acid and the target strand, stringent conditions are almost unacceptable. Such conditions are well known to those skilled in the art and are preferred for applications requiring high selectivity. Non-limiting applications include isolating nucleic acids (eg, genes or nucleic acid segments thereof) or detecting at least one specific mRNA transcript or nucleic acid segment thereof.

  Stringent conditions may include low salt and / or high temperature conditions (eg, conditions as provided by about 0.02 M to about 0.15 M NaCl at a temperature of about 50 ° C. to about 70 ° C.). The temperature and ionic strength of the desired stringency depends on the length of the particular nucleic acid, target sequence length and nucleobase content, nucleic acid charge composition, and formamide, tetramethylammonium chloride or other in the hybridization mixture. It is understood that this is determined in part by the presence or concentration of the solvent.

These ranges, compositions, and conditions for hybridization are shown by way of non-limiting examples only, and the desired stringency for a particular hybridization reaction is often one or more positive controls Or it is understood that it is determined experimentally by comparison with a negative control. Depending on the application envisaged, it is preferred to use hybridization variables to achieve a change in the degree of selectivity of the nucleic acid relative to the target sequence. In a non-limiting example, identification or isolation of related target nucleic acids that do not hybridize to nucleic acids under stringent conditions can be achieved by hybridization at low temperature and / or high ionic strength. For example, moderately stringent conditions can be provided by about 0.1-0.25 M NaCl at a temperature of about 37 ° C to about 55 ° C. Under these conditions, hybridization occurs through the probe sequence and the target sequence is not perfectly complementary and there is a mismatch at one or more sites. In another embodiment, low stringency conditions are provided by about 0.15 M to about 0.9 M salt at a temperature range of about 20 ° C to about 55 ° C. Of course, it is within the purview of those skilled in the art to further adjust the low or high stringency conditions to suit a particular application. For example, in other embodiments, hybridization is performed at a temperature of about 20 ° C. to about 37 ° C. under conditions of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 1.0 mM dithiothreitol. Can be achieved. Other hybridization conditions utilized may include about 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ at a temperature range of about 40 ° C. to about 72 ° C.

  Thus, the nucleotide sequences of this disclosure are used for their ability to selectively form double-stranded molecules with complementary stretches of genes or RNA that provide primers for the amplification of DNA or RNA from tissue. Can be done. Depending on the envisaged application, it is preferred to use hybridization conditions to achieve a change in the degree of selectivity of the probe relative to the target sequence.

  Regardless of the length of the nucleic acid segment coding sequence itself of the present invention, this nucleic acid segment can be promoter, enhancer, polyadenylation signal, additional restriction enzyme sites, multiple cloning sites, etc. so that its overall length can vary considerably. Can be combined with other DNA sequences such as Thus, it can be contemplated that almost any length nucleic acid fragment, preferably having a total length limited by ease of preparation and use in the intended recombinant DNA protocol, can be used.

  For example, a nucleic acid fragment can be prepared that comprises a continuous extension of nucleotides that are identical or complementary to SEQ ID NO: 1, 3, 5, 7, or 14. Nucleic acid fragments for use as DNA transcription templates can also be prepared. These fragments can be short or intermediate lengths such as, for example, about 8 nucleotides, about 10 nucleotides to about 14 nucleotides, or about 15 nucleotides to about 20 nucleotides, and about 35,000 base pairs long About 30,000 base pairs long, about 25,000 base pairs long, about 20,000 base pairs long, about 15,000 base pairs long, about 10,000 base pairs long, or up to about 5,000 base pairs long And a DNA segment having a length of about 1,000 base pairs, a length of about 500 base pairs, a length of about 200 base pairs, a length of about 100 base pairs and a length of about 50 base pairs (listed above) All intermediate lengths of these lengths are also useful, including any range inferred therein and any integer inferred within such a range) Considered.

  For example, in these contexts, “intermediate length” means, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, Means any length between presented ranges, such as 120, 130, 140, 150, 160, 170, 180, 190, 200-500; 500-1,000; 1,000-2,000 2,000 to 3,000; 3,000 to 5,000 0; includes all integers in the range of 5,000 to 10,000, including about 12,001, about 12,002, about 13,001, about 13,002, about 15,000, about 20,000, etc. It is readily understood to include sequences up to.

The various nucleic acid segments can be designed based on a specific nucleic acid sequence and can be of any length. By assigning numerical values to the sequence (eg, 1 for the first residue, 2 for the second residue, etc.), an algorithm can be created that defines all nucleic acid segments:
n to n + y
Where n is an integer from 1 to the last number in the sequence, and y is the length of the nucleic acid segment minus 1, where n + y does not exceed the last number in the sequence. Therefore, in the case of 10-mer, the nucleic acid segment has bases 1 to 10, 2 to 11, 3 to 12. . . It corresponds to. In the case of a 15mer, the nucleic acid segment is represented by bases 1-15, 2-16, 3-17. . . It corresponds to. In the case of a 20mer, the nucleic acid segment has bases 1-20, 2-21, 3-22. . . It corresponds to. In certain embodiments, the nucleic acid segment can be a probe or primer. As used herein, “probe” generally refers to a nucleic acid used in a detection method or composition. As used herein, “primer” generally refers to a nucleic acid used in an extension or amplification method or composition.

  Use of hybridization probes between 17-100 nucleotides in length, or in some aspects of the invention, allows the formation of stable and selective duplex molecules up to 1-2 Kb or more in length. . In order to increase the stability and selectivity of the hybrid, molecules having complementary sequences over an extension longer than 20 bases are generally preferred, thereby improving the quality and extent of a particular hybrid molecule. If desired, it is generally preferred to design nucleic acid molecules having complementary sequences spanning 20-30 nucleotides or longer extensions. Such fragments can be readily prepared, for example, by direct synthesis of the fragments by chemical means or introduction of selected sequences into a recombinant vector for recombinant production.

In general, the hybridization probes described herein use a solid phase as a reagent for detection of the expression of the corresponding gene, as in PCR ^TM , in solution hybridization. Useful in both embodiments. In embodiments that include a solid phase, the test DNA (or RNA) is adsorbed or otherwise immobilized on a selected matrix or surface. This immobilized single stranded nucleic acid is then subjected to hybridization with the selected probe under the desired conditions. The conditions selected will depend on the particular environment based on the particular criteria required (eg, depending on G + C content, target nucleic acid type, nucleic acid source, hybridization probe size, etc.). After washing the hybridized surface to remove non-specifically bound probe molecules, hybridization is detected or quantified by the label.

(B. Nucleic acid amplification)
Nucleic acids used as templates for amplification are isolated from cells contained in a biological sample according to standard methodologies (Sambrook et al., 1989). The nucleic acid can be genomic DNA or fractionated cellular RNA or total cellular RNA. If RNA is used, it may be desirable to convert the RNA to complementary DNA. In one embodiment, the RNA is total cellular RNA and is used directly as a template for amplification.

  A primer pair that selectively hybridizes to a nucleic acid is contacted with the isolated nucleic acid under conditions that allow selective hybridization. As defined herein, the term “primer” is meant to encompass any nucleic acid capable of priming the initial nucleic acid synthesis in a template dependent process. Typically, the primers are 10-20 or 30 base pair long oligonucleotides, although longer sequences can be used. Primers can be provided in double-stranded or single-stranded form, but single-stranded form is preferred.

  Once hybridized, the nucleic acid: primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple amplifications are also referred to as “cycles” and are performed until a sufficient amount of amplification product is produced.

  Next, the amplification product is detected. In certain applications, detection can be performed by visual means. Alternatively, detection may include indirect identification of the product via chemiluminescence, radioactive scintillation of incorporated radiolabels or fluorescent labels, or systems using electrical or thermal pulse signals (Affymax technology).

A number of template dependent processes are available for amplifying marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as RCR ^™ ), which is described in US Pat. Nos. 4,683,195, 4,683,202 and 4th. , 800,159, each of which is incorporated herein in its entirety.

Briefly, RCR ^™ prepares two primer sequences that are complementary to regions on opposite complementary strands of the marker sequence. Excess deoxynucleoside triphosphate is added to the reaction mixture along with a DNA polymerase (eg, Taq polymerase). If a marker sequence is present in the sample, a primer binds to the marker and the polymerase extends the primer along the marker sequence by addition onto the nucleotide. By increasing and decreasing the temperature of the reaction mixture, the extended primer dissociates from the marker to form a reaction product, excess primer binds to the marker and reaction product, and the process is repeated.

A reverse transcriptase RCR ^™ amplification means can be performed to quantify the amount of mRNA amplified. Methods for reverse transcription of RNA into cDNA are well known and are described in Sambrook et al., 1989. An alternative method for reverse transcription utilizes an RNA-dependent DNA polymerase that is thermostable. These methods are described in WO 90/07641 (filed December 21, 1990), which is incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”), disclosed in EPA 320308, which is incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair binds to the opposite complementary strand of the target such that they are adjacent. In the presence of ligase, the two probe pairs are linked to form a single unit. By a temperature cycle such as RCR ^™ , the bound binding unit is dissociated from the target and then serves as a “target sequence” for binding of excess probe pairs. US Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

  Qβ replicase is described in PCT Application No. PCT / US87 / 00880 (incorporated herein by reference) and can also be used as yet another amplification method in the present invention. In this method, a replicative sequence of RNA having a region complementary to the region of the target sequence is added to the sample in the presence of RNA polymerase. This polymerase copies the replicative sequence and can then be detected.

  Isothermal amplification methods that use restriction endonucleases and ligases to achieve amplification of target molecules that contain the nucleotide 5 '-[α-thio] -triphosphate in one strand of the restriction site are also described in the present invention. Can be useful in amplification.

  Strand Displacement Amplification (SDA) is another method of performing isothermal amplification of nucleic acids, including multiple rounds of strand displacement and synthesis (ie, nick translation). A similar method called Repair Chain Reaction (RCR) involves the annealing of several probes across the region targeted for amplification, followed by a repair reaction, where four of the four bases are There are only two. The other two bases can be added as biotinylated derivatives to facilitate detection. A similar approach is used in SDA. Target specific sequences can also be detected using cyclic probe reaction (CPR). In CPR, a probe having 3 'and 5' sequences of non-specific DNA and an intermediate sequence of specific RNA is hybridized to DNA present in a sample. Upon hybridization, the reaction is treated with RNase H and the product of the probe is identified as a unique product that is released after digestion.

  Yet another amplification method described in UK Patent Application No. 2202328, and PCT Application No. PCT / US89 / 01025, each of which is incorporated herein by reference in its entirety, is used in accordance with the present invention. Can be done. In the former application, “modified” primers are used in PCR-like synthesis, template synthesis and enzyme-dependent synthesis. The primer can be modified by labeling with a capture moiety (eg, biotin) and / or a detection moiety (eg, an enzyme). In the latter application, excess labeled probe is added to the sample. In the presence of the target sequence, the probe binds and is cleaved enzymatically. After cleavage, the target sequence is completely released and bound by excess probe. Cleavage of the labeled probe indicates the presence of the target sequence.

  Other nucleic acid amplification procedures include transcription-based amplification system (TAS) (nucleic acid sequence based amplification (NASBA) and 3SR (Gingeras et al. 10315 (incorporated herein by reference))). In NASBA, nucleic acids are for amplification by standard phenol / chloroform extraction, thermal denaturation of clinical samples, treatment with lysis buffers and minispin columns for DNA and RNA isolation, or RNA extraction with guanidinium chloride. Can be prepared. These amplification techniques involve the annealing of primers with target specific sequences. After polymerization, DNA / RNA hybrids are digested with RNase H, while double-stranded DNA molecules are heat denatured again. In either case, the single stranded DNA is made completely double stranded by the addition of a second target specific primer followed by polymerization. This double stranded DNA molecule is then transcribed in a complex manner by an RNA polymerase such as T7 or SP6. In an isothermal cycling reaction, RNA is reverse transcribed into single stranded DNA, then converted into double stranded DNA, and then transcribed again with an RNA polymerase such as T7 or SP6. The resulting product exhibits a target specific sequence, whether truncated or complete.

  Davey et al., EPA 329 822, which is incorporated herein by reference in its entirety, synthesizes single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA) by cycling. Nucleic acid amplification processes are disclosed that can be used in accordance with the present invention. ssRNA is a template for the first primer oligonucleotide, which is extended by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA: RNA duplex by the action of ribonuclease H (RNase H (RNase specific for RNA in the duplex with either DNA or RNA)). The resulting ssDNA is a template for the second primer, which also contains the sequence of the RNA polymerase promoter (exemplified by T7 RNA polymerase) 5 'to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large “Klenow” fragment of E. coli DNA polymerase I), having a sequence identical to the sequence of the original RNA between the primers and a promoter. A double-stranded DNA (“dsDNA”) molecule is obtained that further has a sequence at one end. This promoter sequence can be used by an appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle resulting in very fast amplification. With proper selection of the enzyme, this amplification can be performed isothermally without adding the enzyme in each cycle. Due to the cycling nature of this process, the starting sequence can be selected to be in the form of either DNA or RNA.

  Miller et al., PCT application WO 89/06700 (incorporated herein by reference in its entirety) describes the hybridization of a promoter / primer sequence to a target single stranded DNA (“ssDNA”) followed by this sequence. Nucleic acid sequence amplification schemes based on the transcription of many RNA copies of are disclosed. This scheme is not a cycle. That is, no new template is generated from the resulting RNA transcript. Other amplification methods include “RACE” and “one-sided PCR” (Frohman, 1990, incorporated herein by reference).

  A method based on the ligation of two (or more) oligonucleotides in the presence of a nucleic acid having the resulting “di-oligonucleotide” sequence thereby amplifying the di-oligonucleotide is also an amplification step of the invention. Can be used.

(C. Nucleic acid detection)
In certain embodiments, the nucleic acid sequences of the invention (eg, SEQ ID NO: 1, 3, 5, 7, 14, or all or part of these variants) are used for hybridization assays, RNase protection, and Northern hybridization. It is advantageous to use in combination with any suitable means (eg, label). A wide variety of suitable indicator means are known in the art and include fluorescent ligands, radioactive ligands, enzyme ligands or other ligands that can be detected (eg, avidin / biotin). In preferred embodiments, it may be desirable to use fluorescent labels or enzyme tags (eg, urease, alkaline phosphatase or peroxidase) in place of radioactive reagents or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be used to provide a human eye or spectrophotometrically visible detection means for identifying specific hybridization with a sample containing complementary nucleic acids.

  In embodiments where the nucleic acid is amplified, it may be desirable to separate the amplification product from the template and excess primer in order to determine whether specific amplification has occurred. In one embodiment, the amplification products are separated by agarose gel electrophoresis, agarose-acrylamide gel electrophoresis or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., 1989).

  Alternatively, separation can be effected using chromatographic techniques. There are many types of chromatography that can be used in the present invention: adsorption chromatography, partition chromatography, ion exchange chromatography and molecular sieve chromatography, and many specialized techniques for using them (column chromatography, paper Including chromatography, thin layer chromatography and gas chromatography).

  The amplification product must be visualized to confirm amplification of the marker sequence. One representative visualization method involves staining of the gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification product is completely labeled with radiolabeled or fluorometric labeled nucleotides, the amplification product can be exposed to X-ray film after separation or visualized under an appropriate stimulus spectrum.

  In one embodiment, visualization is achieved indirectly. After separation of the amplification products, the labeled nucleic acid probe is contacted with the amplified marker sequence. The probe is preferably conjugated to a chromophore but can be radiolabeled. In another embodiment, the probe is conjugated to a binding partner (eg, antibody or biotin) and the other member of the binding pair carries a detectable moiety.

  In one embodiment, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those skilled in the art and can be found in many standard books on molecular protocols (see Sambrook et al., 1989). Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane (eg, nitrocellulose) to allow transfer and non-covalent binding of nucleic acids. Subsequently, the membrane is incubated with a chromophore-conjugated probe that can hybridize with the target amplification product. Detection is by exposure of this membrane to an X-ray film or ion radiation detection device.

  One example of the above is described in US Pat. No. 5,279,721, incorporated herein by reference. US Pat. No. 5,279,721 discloses an apparatus and method for automated electrophoresis and transfer of nucleic acids. This device allows electrophoresis and blotting without external manipulation of the gel and is ideally suited for carrying out the method according to the invention.

  Other methods for genetic screening to accurately detect mutations in genomic DNA samples, cDNA samples or RNA samples can be used depending on the particular situation.

Historically, point mutations have been detected using a number of different methods, including denaturing gradient gel electrophoresis ("DGGE"), restriction enzyme polymorphism analysis, chemical and enzymatic cleavage methods, and the like. More general procedures currently in use include direct sequencing of target regions amplified by PCR ^™ (see above) and single-stranded conformation polymorphism analysis (“SSCP”). .

  Another method for screening point mutations is based on RNase cleavage of base pair mismatches in RNA / DNA heteroduplex and RNA / RNA heteroduplex. As used herein, the term “mismatch” refers to one or more unpaired nucleotides or errors in a double-stranded RNA / RNA molecule, double-stranded RNA / DNA molecule, or double-stranded DNA / DNA molecule. Defined as the region of paired nucleotides. Thus, this definition includes insertion / deletion mutations as well as mismatches due to single and multiple base point mutations.

  US Pat. No. 4,946,773 includes annealing a single-stranded DNA test sample or single-stranded RNA test sample to an RNA probe and subsequent treatment of the nucleic acid duplex with RNase A. An RNase A mismatch cleavage assay is described. After the RNase cleavage reaction, the RNase is inactivated by proteolytic digestion and organic extraction, and the cleavage product is denatured by heating and analyzed by electrophoresis on a denaturing polyacrylamide gel. For mismatch detection, RNase A treated single stranded products separated by size by electrophoresis are compared against a similarly treated control duplex. Samples containing smaller fragments (cleavage products) that are not found in the control duplex are scored as positive.

  Currently available RNase mismatch cleavage assays (including assays performed according to US Pat. No. 4,946,773) require the use of radiolabeled RNA probes. Myers and Maniatis describe the detection of base pair mismatches using RNase A in US Pat. No. 4,946,773. Other investigators have reported that E. The use of the E. coli enzyme RNase I was described. Since it has a broader cleavage specificity than RNase A, RNase I is a base pair mismatch if the component can be found to reduce the degree of non-specific cleavage and increase the mismatch cleavage frequency. It is a desirable enzyme for use in the detection of. The use of RNase I for mismatch detection is described in documents from Promega Biotech. Promega sells a kit containing RNase I which is shown in its document to cleave three of the four known discrepancies if the enzyme level is high enough.

  The RNase protection assay was first used to detect and map the ends of specific mRNA targets in solution. This assay relies on the ability to readily generate highly specific active radiolabeled RNA probes complementary to the mRNA of interest by in vitro transcription. Originally, the template for in vitro transcription was a recombinant plasmid containing a bacteriophage promoter. Probes are mixed with the total cellular RNA sample to allow hybridization to their complementary target, and then the mixture is treated with RNase to degrade excess unhybridized probe. Also, as originally intended, the RNase used is specific for single stranded RNA, so that the hybridized double stranded probe is protected from degradation. After incubation and removal of RNase, the protected probe (which is proportional to the amount of target mRNA present) is recovered and analyzed on a polyacrylamide gel.

The RNase protection assay was adapted to detect single base mutations. In this type of RNase A mismatch cleavage assay, a radiolabeled RNA probe transcribed in vitro from a wild type sequence hybridizes to a complementary target region derived from a test sample. Test targets generally include DNA (either genomic DNA or DNA cloned by plasmids or amplified by PCR ^™ ), but RNA targets (endogenous mRNA) are sometimes used. If one (or more) nucleotide sequence difference occurs between the hybridized probe and target, the resulting disruption in Watson-Crick hydrogen bonding at that position ("mismatch") is in some cases single stranded It can be recognized and cleaved by a specific ribonuclease. To date, RNase A has been used almost exclusively for single base mismatch cleavage, while RNase I has recently been shown to be useful for mismatch cleavage. There have been recent descriptions of using MutS protein and other DNA repair enzymes for the detection of single base mismatches.

  RNA can be measured using nuclease S1 analysis of the reaction products. An exemplary procedure for S1 analysis is as follows: RNA of interest (0.005 to .0. 0) in S1 hybridization buffer (80% formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM Pipes, pH 6.4). 1 mg) and an excess of S1 probe containing a labeled oligonucleotide complementary to 20-80 or more consecutive nucleotides of specific RNA. After denaturation at 94 ° C. for 4 minutes, overnight hybridization at 30 ° C. and precipitation with ethanol, the S1 probe / RNA mixture was washed with S1 buffer (0.26 M NaCl, 0.05 M sodium acetate, pH 4.6). ) And 4.5 mM zinc sulfate). The sample is divided into two volumes and 100 units of S1 nuclease (Sigma Chemical Company) is added to one tube. Samples are incubated at 37 ° C. for 60 minutes; EDTA (10 mM final concentration) and 15 g poly I-poly C RNA are then added and the samples are extracted with phenol / chloroform and precipitated in ethanol. The sample is then subjected to polyacrylamide gel electrophoresis.

One method of producing radiolabeled RNA probes with high specific activity involves mixing radiolabeled NTPs during transcription. Suitable isotopes for radiolabeling include ³⁵ S labeled UTP, GTP, CTP or ATP and ³² P labeled UTP, GTP, CTP or ATP. For optimal results, a gel-purified radiolabeled RNA probe with a specific activity of 1-3 × 10 ⁸ cpm / μg, mainly 300-500 bases long, is made using the RNA polymerase of the present invention Should. It is often advisable to use NTPs with high specific activity (eg, 3000 Ci / mmol [α- ³² P] CTP) to generate this in vitro transcript. To prevent background hybridization, it is important to remove plasmid template DNA, for example, by digestion that can be performed using RQ1 RNase-Free DNase, followed by phenol: chloroform: isoamyl alcohol extraction and ethanol precipitation. .

  Alternative methods for making radiolabeled probes include the use of riboprobe systems, radiolabeled RNA probes, or microgram quantities of in vitro transcripts that can produce a high degree of specific activity. Riboprobes are useful with radiolabeled RNA probes in many applications, including RNase protection, Northern hybridization, S1 analysis, and in situ hybridization assays. The essential components of an in vitro transcript are a riboprobe, an RNA polymerase, a DNA template containing a phage RNA polymerase promoter, and a ribonucleotide triphosphate.

(D. Cloning of vRNAP gene)
The present invention contemplates cloning vRNAP, or more specifically a mini-vRNAP gene. Today, techniques often used by those skilled in the art for protein production include obtaining a so-called “recombinant” form of a protein, expressing the protein in a recombinant cell, and from such a cell, Obtaining a polypeptide or peptide. These techniques are based on the “cloning” of nucleic acid molecules encoding proteins from a DNA library, ie obtaining a specific DNA molecule that is different from other parts of the DNA. This can be achieved, for example, by cloning of cDNA molecules or cloning of genomic-like DNA molecules.

  The first step in such a cloning procedure is the screening of an appropriate DNA library (eg, phage, bacteria, yeast, fungus, mouse, rat, monkey, or human). This screening protocol may utilize nucleotide segments or probes designed to hybridize to prokaryotic-derived vRNAP cDNA or gene sequences. In addition, using an antibody designed to bind to the expressed vRNAP protein, polypeptide or peptide as a probe, a suitable viral DNA expression library, eubacterial DNA expression library, archaeal DNA expression library, Alternatively, eukaryotic cell DNA expression libraries can be screened. Alternatively, activity assays can be used. The operation of such screening protocols is well known to those skilled in the art and is described in detail in the scientific literature (eg, Sambrook et al. (1989), incorporated herein by reference). Furthermore, since the present invention encompasses cloning gene segments and cDNA molecules, it is contemplated that suitable gene cloning methods known to those skilled in the art can also be used.

  A relatively small peptide (eg, from about 8 amino acids in length to about 9 amino acids in length, about 10 amino acids in length, as described in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 15) About 11 amino acids long, about 12 amino acids long, about 13 amino acids long, about 14 amino acids long, about 15 amino acids long, about 16 amino acids long, about 17 amino acids long, about 18 amino acids long, about 19 amino acids long, about 20 amino acids long, About 21 amino acids long, about 22 amino acids long, about 23 amino acids long, about 24 amino acids long, about 25 amino acids long, about 26 amino acids long, about 27 amino acids long, about 28 amino acids long, about 29 amino acids long, about 30 amino acids long, About 31 amino acids, about 32 amino acids, about 33 amino acids, about 34 amino acids, about 35 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids Acid length, peptides up to about 50 amino acids in length, and more preferably peptides from about 15 amino acids in length to about 30 amino acids in length), and proteins corresponding to the full-length sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 15 Also encompassed by the present invention are DNA segments that encode larger polypeptides, as well as larger polypeptides comprising those proteins, and any range that can be drawn within, and any integer number of peptides that can be drawn within that range. Furthermore, in addition to “standard” DNA and RNA nucleotide bases, modified bases are also contemplated for use in certain applications of the invention. A table of exemplary modified bases is provided herein below, but is not limited thereto.

（ＩＩＩ．組換えベクター、プロモーター、宿主細胞および発現）
組換えベクターは、本発明の重要なさらなる局面を形成する。用語「発現ベクターまたは構築物」とは、遺伝子産物をコードする核酸を含む、任意の型の遺伝子構築物を意味し、ここで、配列をコードする一部または全部の核酸が転写され得る。この転写物は、タンパク質性の分子に翻訳され得るが、１本鎖ＤＮＡテンプレートを使用してＲＮＡを転写するミニｖＲＮＡＰの場合のように、転写物が翻訳される必要はない。従って、特定の実施形態において、発現は、１本鎖ＤＮＡの転写およびＲＮＡのタンパク質産物への翻訳の両方を含む。他の実施形態において、発現は核酸の転写のみを含む。組換えベクターもまた、本発明のＲＮＡの送達のために使用され得る。

(III. Recombinant vectors, promoters, host cells and expression)
Recombinant vectors form an important further aspect of the present invention. The term “expression vector or construct” refers to any type of gene construct, including nucleic acid encoding a gene product, wherein some or all of the nucleic acid encoding the sequence can be transcribed. This transcript can be translated into a proteinaceous molecule, but the transcript need not be translated as in the case of mini-vRNAP that transcribes RNA using a single-stranded DNA template. Thus, in certain embodiments, expression includes both transcription of single stranded DNA and translation of RNA into a protein product. In other embodiments, expression includes transcription of the nucleic acid only. Recombinant vectors can also be used for delivery of the RNA of the invention.

  Particularly useful vectors are contemplated in which the coding portion of the DNA segment is placed under the transcriptional control of a promoter, whether it translates a full-length protein or a smaller polypeptide or peptide. “Promoter” refers to a DNA sequence recognized by, or introduced into, a synthesis mechanism of a cell that is required to initiate specific expression of a gene. The phrases “operably located”, “under control” or “under transcriptional control” indicate that the promoter is in the correct position and orientation relative to the nucleic acid that controls RNA polymerase initiation and gene expression. means.

  One specific useful vector is pBAD. This pBAD expression vector allows greater control of bacterial expression of the recombinant protein and allows tight regulation to switch expression on or off. pBAD vectors allow dose-dependent induction of regulation of expression levels. The pBAD expression system is one of the two most common problems of heterologous protein expression in bacteria: the toxicity of the recombinant protein to the host, and the case where the recombinant protein is expressed at a highly uncontrolled level. It helps to overcome the insolubility of the replacement protein. In both cases, a tightly regulated expression system is important to maximize the yield of the recombinant protein. This pBAD expression system is E. coli. Based on the araBAD operon that controls the arabinose metabolic pathway in E. coli and allows for precise control of heterologous expression to levels that are optimal for recovering the protein of interest in high yield (Guzman et al., 1995).

(A. Promoter)
Any promoter normally found in a native state host cell is used in the present invention to drive the expression of N4 vRNA polymerase, or mini-vRNA polymerase. Alternatively, an abnormal promoter found in a native state host cell that is recognized by a natural, normal natural host cell RNA polymerase, or a non-natural RNA polymerase expressed intracellularly, is an expression of RNA polymerase. Can be used herein to drive. Other promoters are selected from nucleic acid sequence databases accessible to those skilled in the art (eg, GenBank), or promoters can be isolated by screening methods. A promoter recognized by the host cell can be operably linked to a gene (or group of genes) encoding N4 RNA polymerase. This operable linkage can be constructed using any known technique for DNA manipulation, as referred to herein.

  Promoters are described as either constitutive or inducible. Constitutive promoters actively drive the expression of genes under their control. In contrast, inducible promoters are activated in response to specific environmental stimuli. Both constitutive and inducible promoters can be used in the present invention to express non-host genes in host cells.

  Inducible promoters include, but are not limited to, trp, tac, lac, ara, recA, λPr and λPl. The host cell is E. coli. In embodiments that are E. coli, these promoters and other promoters that can be used in the present invention for expression of N4 vRNA polymerase or mini-vRNA polymerase are Makrides, Microbiological Reviews (1996), 60, 512-538 (this Which is incorporated herein by reference). Further, the host cell is E. coli. In embodiments of the invention that are other microorganisms other than E. coli (eg, Saccharomyces, Bacillus, and Pseudomonas), any inducible promoter known to those skilled in the art to be active in the host cell is used. Can drive the expression of heterologous RNA polymerase (US Pat. No. 6,218,145).

In combination with the compositions disclosed herein, for example, recombinant cloning and / or PCR ^™ technology (PCR ^™ technology is described in US Pat. No. 4,863,202 and US Pat. No. 4,682,682). Can be obtained by isolating a 5 ′ untranslated region located upstream of a coding segment or exon using No. 195, each of which is disclosed herein by reference. The promoter may be in the form of a promoter that is naturally associated with N4 vRNA polymerase or mini vRNA polymerase.

  In other embodiments, it is contemplated to obtain certain advantages by placing the coding DNA segment under the control of a recombinant or heterologous promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with N4 vRNA polymerase or mini-vRNA polymerase in its natural environment. Such promoters include promoters normally associated with other genes, and / or promoters isolated from any other bacteria, virus, eukaryotic cell, protist cell, or mammalian cell, and And / or non-naturally occurring promoters made by human hand (ie, including different elements from different promoters, or mutations that increase, decrease or alter expression).

  In nature, it is important to use a promoter that is selected for expression and that effectively directs the expression of the DNA segment in the cell type, organism or even mammal. The use of a combination of promoter and cell type for protein expression is generally known to those skilled in the field of molecular biology (see, eg, Sambrook et al. (1989), which is described herein). Incorporated by reference in the book)). The promoter used can be constitutive or inducible and can be used under appropriate conditions (eg recombinant protein, recombinant polymorphism) to direct high level expression of the introduced DNA segment. As advantageous in large-scale production of peptides or recombinant peptides).

  At least one module in the promoter generally functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but overlays the start site itself in promoters that lack some TATA boxes (eg, promoters of mammalian terminal deoxynucleotide transferase genes and SV40 late genes). The decentralized element helps to fix the starting position.

  Additional promoter elements regulate the frequency of transcription initiation. Many promoters have been shown to contain functional elements also downstream of the start site, but typically these promoter elements are located in the region 30-110 bp upstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is preserved even when elements are swapped or moved relative to each other. In the thymidine kinase promoter, the spacing between promoter elements can increase to 50 base pairs before activity begins to decrease. Depending on the promoter, the individual elements appear to be able to function either cooperatively or independently to activate transcription.

  The particular promoter used to control the expression of the nucleic acid is believed to be unimportant as long as the nucleic acid can be expressed in the targeted cell. Thus, when targeting human cells, it is preferred to place the nucleic acid coding region adjacent to or under the control of a promoter that can be most expressed in human cells. Generally speaking, such promoters can include either human or viral promoters.

  In various other embodiments, the human cytomegalovirus (CMV) immediate early promoter, the SV40 immediate early promoter, and the rous sarcoma virus long terminal repeat can be used to obtain rapid high-level expression of nucleic acids. The use of other viral promoters, mammalian intracellular promoters or bacterial phage promoters known in the art to achieve expression, where the level of expression is sufficient for a given purpose, is also contemplated. Tables 3 and 4 below list several elements / promoters that can be used to regulate the expression of the vRNAP gene in the context of the present invention. This list is not intended to be exhaustive of all elements that may be associated with enhanced expression, but merely as an illustration of these.

In certain embodiments of the invention, promoter sequences that are specifically recognized by DNA-dependent RNA polymerases (eg, but are not limited to those described by Chamberlin and Ryan (1982), and Jorgensen et al. (1991)). ) Can be used. These promoters can be used to express wild-type or mutant forms of the mini-V RNA polymerase of the invention. Several RNA polymerase promoter sequences are useful in nature, including promoters derived from SP6 (eg, Zhou and Doetsch, 1993), promoters derived from T7 (eg, Martin and Coleman, 1987), and T3 Promoters derived from (eg, McGraw et al., 1985), but are not limited thereto. RNA polymerase promoter sequences from Thermus thermophilus can also be used (see, eg, Wendt et al., 1990; Faraldo et al., 1992; Hartmann et al., 1987; Hartmann et al., 1991). The length of the promoter sequence varies depending on the promoter selected. For example, the T7 RNA polymerase promoter is only about 25 bases long and acts as a functional promoter, while other promoter sequences require more than 50 bases to provide a functional promoter. In other embodiments, a promoter that is recognized by an RNA polymerase from a T7-like bacteriophage is used. The gene structure of all T7-like bacteriophages tested was found to be essentially the same as the T7 gene structure. Examples of T7-like bacteriophages according to the present invention include Escherichia coli phage T3, phage phiI, phage phiII, phage W31, phage H, phage Y, phage A1, phage 122, phage cro, phage C21, phage C22, and phage Phage gh-1 of Pseudomonas putida; Phage SP6 of Salmonella typhimurium; Phage IV of Serratia marcescens; Phage ViIII of Citrobacter; and Phage No. of Klebsiella. 11 (Hausmann, 1976; Korsen et al., 1975; Dunn et al., 1971; Towle et al., 1975; Butler and Chanberlin, 1982).

  If a T7 RNA polymerase promoter or another T7-like RNA polymerase promoter is used to express the wild-type or mutant form of the gene of the mini-V RNA polymerase of the invention, the T7 RNA polymerase, or the promoter used The gene can be expressed in a host cell that expresses a corresponding T7-like RNA polymerase for, where the RNA polymerase for that promoter is either constitutively expressed or preferably from an inducible promoter. Expressed in For illustrative purposes, the T7 RNA polymerase expression system is described, for example, in US Pat. Nos. 5,693,489 and 5,869,320, the disclosures of which are hereby incorporated by reference in their entirety. However, the present invention is not limited to such an expression system.

(B. Enhancer)
Enhancers were originally detected as genetic elements that increase transcription from remotely located promoters on the same DNA molecule. The ability of this enhancer to act over large distances has little precedent in classical studies of prokaryotic transcriptional regulation. Subsequent studies have shown that the DNA region with enhancer activity is very similar in structure to the promoter. That is, these DNA regions are composed of many individual elements, each of which binds to one or more transcription proteins.

  The basic difference between enhancers and promoters is operability. The entire enhancer region must be able to stimulate transcription remotely. This need not be true for the promoter region or its component elements. On the other hand, a promoter must have one or more elements that are directed at initiating RNA synthesis at a specific site and in a specific direction, while enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often appearing to have a very similar modular organization.

  In addition, any promoter / enhancer combination (Eukaryotic Promoter Data Base EPDB, http://www.epd.isb-sib.ch/) can also be used to drive expression. Eukaryotic cells, if appropriate bacterial polymerases are provided, support intracytoplasmic transcription from specific bacterial promoters, either as part of a delivery complex or as an additional gene expression construct. obtain.

本発明のｖＲＮＡＰ、ミニｖＲＮＡＰまたはそれらの変異体を使用した転写後のタンパク質性分子の発現を変化させて、一旦適切なクローン（またはクローン群）を獲得すると、これらがｃＤＮＡベースであれゲノムベースであれ、発現系を調製し得る。原核細胞系または真核細胞系における発現のためにＤＮＡセグメントを操作することは、組換え発現の当業者に一般的に公知の技術によって達成し得る。実質的にはあらゆる発現系が本発明のタンパク質性分子の発現において使用され得ると考えられる。

Once the expression of protein molecules after transcription using the vRNAP, mini-vRNAP or variants thereof of the present invention is altered to obtain appropriate clones (or groups of clones), these can be either cDNA-based or genomic-based. Anyway, an expression system can be prepared. Manipulating DNA segments for expression in prokaryotic or eukaryotic cell systems can be accomplished by techniques generally known to those skilled in the art of recombinant expression. It is contemplated that virtually any expression system can be used in the expression of the proteinaceous molecule of the present invention.

  Both cDNA and genomic sequences are suitable for expression in eukaryotes. This is because host cells generally process genomic transcripts and produce functional mRNAs for translation into proteinaceous molecules. In general, it may be more convenient to use a cDNA version of a gene as a recombinant gene. The use of a cDNA version is generally larger than a genomic gene (typically up to an order of magnitude more than a cDNA gene) in order to transfect targeted cells, It offers an advantage in that it is even smaller and easier to use. However, it is contemplated that a genomic version of a particular gene can be used if desired.

  In expression, a gene typically includes a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not considered important in the successful implementation of the invention, and any such sequence can be used. Preferred embodiments include an SV40 polyadenylation signal and a bovine growth hormone polyadenylation signal that are convenient and known to function well in a variety of target cells. A terminator is also contemplated as an element of the expression cassette. These elements can help to enhance message levels and minimize reading from cassettes to other sequences.

(C. Antisense and ribozymes)
In some embodiments of the invention, vRNA polymerase can be used to synthesize antisense RNA or ribozymes.

  The term “antisense nucleic acid” is intended to refer to an oligonucleotide that is complementary to the base sequence of DNA and RNA. Antisense oligonucleotides, when transduced into target cells, specifically bind to their target nucleic acids and interfere with transcription, RNA processing, transport, translation, and / or stability. Targeting double stranded (ds) DNA with oligonucleotides leads to triple helix formation; RNA targeting leads to double helix formation. The antisense nucleic acid can be complementary to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 14, can be complementary to a mini-vRNAP coding sequence, or a mini-vRNAP non-coding sequence. Can be complementary. Antisense RNA constructs or DNA encoding such antisense RNA can be used, either gene transcription or translation in a host cell, either in vitro or in vivo (eg, within a host animal including a human subject). Or inhibit both.

  Antisense constructs can be designed to bind to promoters and other regulatory regions, gene exons, gene introns or even exon-intron boundaries (splice sites) of genes. It is contemplated that the most effective antisense constructs can include a region complementary to the intron / exon splice site. Thus, antisense constructs with complementary regions within 50-200 bases of intron / exon splice sites can be used. It has been observed that some exon sequences can be included in constructs that do not significantly affect their target selectivity. The amount of exonic material included will vary depending on the particular exon and intron sequences used. Whether it contains so much exon DNA is easily tested by simply testing the construct in vitro, whether normal cellular function is affected, or the expression of related genes with complementary sequences. It can be determined whether it is affected.

  As described above, “complementary” or “antisense” refers to polynucleotide sequences that are substantially complementary over their entire length and have only a few base mismatches. For example, sequences that are 15 bases long can be said to be complementary if they have nucleotides complementary to positions 13 or 14. Of course, sequences that are completely complementary are sequences that are completely complementary over their entire length and have no base mismatch. Other sequences with a low degree of homology are also contemplated. For example, antisense constructs can be designed that have limited regions of high homology but also include non-homologous regions (eg, ribozymes). Although these molecules have less than 50% homology, they bind to the target molecule under appropriate conditions.

  It may be advantageous to combine a portion of genomic DNA with a cDNA sequence or a synthetic sequence to produce a specific construct. For example, if an intron is desired in the final construct, a genomic clone needs to be used. A cDNA or synthetic polynucleotide may provide a more convenient restriction site for the rest of the construct and is therefore used for the rest of the sequence.

  All or part of the gene sequence can be used in the background of an antisense construct, statistically any 17 base long sequence will occur only once in the human genome, and thus a single target sequence Should be enough to identify. Although shorter oligomers are created in vivo accessibility and easier to increase, many other factors are involved in determining the specificity of hybridization. Both oligonucleotide binding affinity and sequence specificity for complementary targets increase with increasing length. It is contemplated that 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pair oligonucleotides are used. Whether a given antisense nucleic acid is effective in targeting the corresponding host cell gene is readily determined by simply testing the construct in vivo and whether the function of the endogenous gene is affected or It can be determined whether the expression of related genes with complementary sequences is affected.

  In certain embodiments, it may be desirable to use an antisense construct that includes other elements (eg, elements comprising C-5 propyne pyrimidine). Oligonucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al., 1993).

  As an alternative to targeted antisense delivery, targeted ribozymes can be used. The term “ribozyme” refers to an RNA-based enzyme that can target and cleave specific base sequences in oncogene DNA and RNA. The ribozyme can either be targeted directly into the cell in the form of an RNA oligonucleotide incorporating the ribozyme sequence, or can be introduced into the cell as an expression construct encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same way as described for antisense nucleic acids. The sequence for the ribozyme is included in the DNA template and can remove the 5 'terminal sequence in the RNA generated via T7 RNA polymerase transcription.

  Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have unique catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlac et al., 1987; Forster and Symons, 1987). For example, a vast number of ribozymes accelerates the phosphoester rearrangement reaction with a high degree of specificity and often cleaves only one of several phosphodiester bonds in oligonucleotides (Cech et al., 1981; Michel). And Westoff, 1990; Reinhold-Hurek and Shub, 1992). This specificity is due to the need for the substrate to bind to the ribozyme internal guide sequence ("IGS") via specific base pair interactions prior to chemical reaction.

  Ribozyme catalysis was first observed as part of a sequence-specific cleavage / ligation reaction involving nucleic acids (Joyce, 1989; Cech et al., 1981). For example, US Pat. No. 5,354,855, certain ribozymes can act as endonucleases with a sequence specificity that is higher than the sequence specificity of known ribonucleases and close to that of DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suitable for therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990; Sioud et al., 1992). Recently, ribozymes have been reported to induce genetic changes in some cell lines to which they have been applied, and the modified genes include genes for the oncogenes H-ras, c-fos and HIV. . Most of this work involves the modification of target mRNAs based on specific mutant codons that are cleaved by specific ribozymes. From the information contained herein and the knowledge of those skilled in the art, the preparation and use of additional ribozymes that specifically target a given gene is now straightforward.

  Several different ribozyme motifs are described along with RNA cleavage activity (reviewed in Symons, 1992). Examples of ribozymes include tobacco ring spot virus (Prody et al., 1986), avocado sun blotch viroid (Palukaitis et al., 1979; Symons, 1981), and Lucerne transient streak virus (Forster and Symon, 1987) and other sequences derived from self-splicing intron group I. These and related virus-derived sequences are referred to as hammerhead ribozymes based on the predicted folded secondary structure.

  Other suitable ribozymes include RNaseP with RNA-cleaving activity (Yuan et al., 1992; Yuan and Altman, 1994), hairpin ribozyme structure (Berzal-Herranz et al., 1992; Chowira et al., 1993) and delta hepatitis virus-based ribozymes And sequences derived from (Perrotta and Been, 1992). The general design and optimization of ribozymes directed to RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, 1988; Symmons, 1992; Chokira et al., 1994; and Thompson et al., 1995).

  Another variable in ribozyme design is the selection of the cleavage site on a given target RNA. Ribozymes are targeted to a given sequence by annealing to specific sites through complementary base pair interactions. Two homologous stretches are required for this targeting. These stretches of homologous sequences are adjacent to the catalytic ribozyme structure defined above. Each stretch of homologous sequence can vary from 7 to 15 nucleotides in length. The only requirement for defining this homologous sequence is that in the target RNA these homologous sequences are interrupted by a specific sequence that is a cleavage site. For hammerhead ribozymes, the cleavage site is a dinucleotide on the target RNA (uracil (U) followed by either adenine, cytosine or uracil (A, C or U)) (Perriman et al., 1992; Thompson). Et al., 1995). The frequency with which this dinucleotide is present in any given RNA is statistically 3 out of 16. Thus, for a given target messenger RNA of 1000 bases, 187 dinucleotide cleavage sites can be statistically present.

  Designing and testing ribozymes for efficient cleavage of target RNA is a process well known to those skilled in the art. Examples of specific methods for designing and testing ribozymes are described by Chowira et al. (1994) and Lieber and Strauss (1995), each incorporated by reference. Identification of a working and preferred sequence for use in ribozymes is simply a matter of preparing and testing a given sequence and is a routinely performed “screening” method known to those skilled in the art. It is.

  A specific initiation signal may also be required for efficient translation of the coding sequence. These signals include the ATG start codon and adjacent sequences. Exogenous translational control signals (including the ATG start codon) may need to be provided. One skilled in the art can easily determine this and can easily provide the necessary signal. It is well known that the initiation codon must be “in frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancing elements.

(D. Host cell)
Host cells can be derived from prokaryotes or eukaryotes (including yeast cells), insect cells, and mammalian cells, where the desired result is either replication of the vector or part or all of the nucleic acid sequence encoded by the vector. It depends on whether or not it is expressed. Many cell lines and cultures are available for use as host cells and these can be obtained through the American Type Culture Collection (ATCC). The ATCC is a tissue that serves as a repository for growth cultures and genetic material (www.atcc.org). The appropriate host can be determined by one skilled in the art based on the vector backbone and the desired result. For example, a plasmid or cosmid can be introduced into a prokaryotic host cell for replication of many vector copies. Bacterial cells used as host cells for vector replication and / or expression include DH5α, BL21, JM109, and KC8, as well as many commercially available bacterial hosts (eg, SURE® Competent Cells and SOLOPACK Gold Cells). (STRATAGENE®, La Jolla, CA)). Alternatively, bacterial cells (eg, E. coli LE392) can be used as host cells. Suitable yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris.

  Examples of eukaryotic host cells for vector replication and / or expression include HeLa, NIH3T3, Jurrat, 293, Cos, CHO, Saos, BHK, C127 and PC12. Many host cells from various cell types and organisms are available and known to those skilled in the art. Similarly, viral vectors can be used with either eukaryotic or prokaryotic host cells, particularly host cells that allow replication or expression of the vector.

  Some vectors may use control sequences that allow replication and / or expression in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions that incubate, maintain and allow replication of all of the host cells described above. Techniques and conditions that allow the production of large-scale vectors and the nucleic acids encoded by the vectors and / or their cognate polypeptides, proteins, or peptides are also understood and known.

  vRNAP (ie, more specifically, mini-vRNAP) can be co-expressed with other selected protein-like molecules (eg, EcoSSB and other proteins of interest), where these protein-like molecules are identical It is proposed that the vRNAP gene can be co-expressed in a cell or provided to a cell already having another selected proteinaceous molecule. Co-expression can be achieved by co-transfecting the cells with two different recombinant vectors carrying either of each DNA. Alternatively, a single recombinant vector can be constructed to contain both coding regions of a proteinaceous molecule, which can then be expressed in cells transfected with the single vector. . In any event, the term “co-expression” herein refers to the expression of both the vRNAP gene and other selected proteinaceous molecules in the same recombinant cell.

  As used herein, the terms “genetically engineered” cells and “recombinant” cells or “genetically engineered” host cells and “recombinant” host cells refer to exogenous DNA segments or exogenous genes. A cell into which (for example, a gene encoding cDNA, vRNAP, mini-vRNAP or a variant thereof) has been introduced. Thus, genetically engineered cells can be distinguished from naturally occurring cells that do not contain exogenously introduced exogenous DNA segments or exogenous genes. Thus, genetically engineered cells include cells having introduced cDNA or genomic genes and genes located adjacent to promoters that are not naturally associated with the particular introduced gene.

  In order to express a recombinant vRNAP according to the present invention, a nucleic acid encoding a wild type or a mutant vRNAP protein-like molecule under the control of one or more promoters, whether mutant or wild type. An expression vector containing a nucleic acid encoding is prepared. Transcription of the transcription reading frame between about 1 and about 50 nucleotides “downstream” (ie, 3 ′) of the selected promoter, in order to bring the coding sequence “under control” of the promoter. Place the 5 'end of the start site. An “upstream” promoter directs transcription of the DNA and promotes expression of the encoded recombinant protein, polypeptide or peptide. This is the meaning of “recombinant expression” in this context.

  Many standard techniques are available for constructing expression vectors containing appropriate nucleic acids and transcriptional / translational control sequences for expressing proteins, polypeptides or peptides in a variety of host expression systems. Cell types available for expression include bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA expression vectors, recombinant plasmid DNA expression vectors or recombinant cosmid DNA expression vectors. However, it is not limited to these.

  Specific examples of prokaryotic hosts include E. E. coli RR1 strain, E. coli. E. coli strain LE392, E. coli. E. coli B strain, E. coli E. coli strain X 1776 (ATCC accession number 31537) and E. coli strain X E. coli strain W3110 (F-, λ-, prototrophic strain, ATCC accession number 273325); genus Bacillus (eg, Bacillus subtilis); ).

  In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used with these hosts. This vector usually carries a replication origin and exhibits sequences that can provide phenotypic selection in transformed cells. For example, E.I. E. coli is often transformed using a derivative of pBR322 (a plasmid derived from an E. coli species). pBR322 contains a gene resistant to ampicillin and a gene resistant to tetracycline and thus provides an easy way to identify transformed cells. The pBR plasmid, or other microbial plasmid or phage, can also contain or be modified to contain a promoter that can be used by the microorganism for expression of its own protein.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microbial organism can be used with these hosts as transformation vectors. For example, the phage λ GEM ^™ -11 can be used to make recombinant phage vectors that can be used to transform host cells (eg, E. coli LE392).

  Further useful vectors include the pIN vector (Inouye et al., 1985); and the pGEX vector (for use in the production of glutathione S-transferase (GST) soluble proteins for subsequent purification and separation or cleavage). It is done.

  The following details regarding recombinant protein production in bacterial cells (eg, E. coli) are generally provided by exemplary information on recombinant proteins, and adaptation to a particular recombinant expression system is known to those skilled in the art. It is known.

  Bacterial cells (eg, E. coli) containing the expression vector are grown in any of a number of suitable media (eg, LB). Expression of the recombinant protein-like molecule can be induced by adding IPTG or a suitable inducer to the medium, or by changing the incubation to a higher temperature, depending on the controlled promoter used. After culturing the bacteria for an additional period (generally between 2 and 24 hours), the cells are harvested by centrifugation and washed to remove the remaining medium.

  The bacterial cells are then lysed, eg, by disruption in a cell homogenizer, by sonication, or by a cell press and centrifuged to separate dense inclusion bodies and cell membranes from soluble cell components. . This centrifugation can be performed under conditions where dense inclusion bodies are selectively concentrated by mixing sugar (eg, sucrose) into a buffer and centrifuging at a selective speed.

  When recombinant protein-like molecules are expressed in inclusion bodies, as in many instances, these inclusion bodies are one of several solutions to remove some contaminated host cells. Containing high concentrations of urea (eg 8M) or chaotropic agents (eg guanidine hydrochloride in the presence of reducing agents (eg β-mercaptoethanol or DTT (dithiothreitol))) Solubilized in the solution.

  Under some circumstances, a protein-like molecule is incubated for several hours under conditions suitable to undergo a process of rewinding to a conformation that closely resembles that of the native protein-like molecule That can be more advantageous. Such conditions generally include low protein-like molecular concentrations (less than 500 mg / ml), low levels of reducing agent, less than 2 M urea concentration, and often a mixture of oxidized and reduced glutathione (intraprotein-like molecules The presence of a reagent such as to facilitate mixing of disulfide bonds.

  The refolding process can be performed, for example, by SDS-PAGE or using antibodies specific for native molecules (which can be obtained from animals vaccinated with native molecules or smaller amounts of recombinant protein-like molecules). Can be monitored. After refolding, the proteinaceous molecule is further purified and the refolded mixture by chromatography on any of several supports (including ion exchange resins, gel filtration resins) or on various affinity columns. Can be separated from

  For expression in Saccharomyces, for example, the plasmid YRp7 is commonly used. This plasmid already contains the trp1 gene that provides a selectable marker for mutant yeast strains (ATCC accession number 44076 or PEP4-1) that lack the ability to increase tryptophan. Therefore, the presence of trp1 damage as a property of the yeast host genome provides an effective environment for detecting transformation by growth in the absence of tryptophan.

  Suitable promoter sequences in yeast vectors include 3-phosphoglycerate kinase or other glycolytic enzymes (eg, enolase, glyceraldehyde-3-phosphate protein, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose -6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase). In the construction of appropriate expression plasmids, the termination sequences associated with these genes are also ligated into an expression vector 3 'of the desired sequence to be expressed providing mRNA polyadenylation and termination.

  In addition to microorganisms, cell cultures derived from multicellular organisms can also be used as hosts. In principle, any such cell culture can work, whether it is a vertebrate culture or a non-vertebrate culture. In addition to mammalian cells, these cells include insect cell lines infected with recombinant viral expression vectors (eg, baculovirus); and recombinant viral expression vectors (eg, cauliflower mosaic virus (CaMV); tobacco mosaic virus). Plant cell lines infected with (TMV)) or plant cell lines transformed with a recombinant plasmid expression vector (eg Ti plasmid) comprising one or more RNAP coding sequences.

  Different host cells have characteristic and specific mechanisms for post-transcriptional processing and protein-like molecule modification. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein-like molecule expressed.

  Many virus-based expression systems are used, and commonly used promoters are derived from polyoma virus, adenovirus 2, and most often simian virus 40 (SV40). The early and late promoters of the SV40 virus are particularly useful. Because both are easily obtained from the virus as a fragment that also contains the SV40 virus origin of replication. A smaller or larger SV40 fragment can also be used if it contains an approximately 250 bp sequence extending from the HindIII site to the BglI site located at the viral origin of replication.

  In cases where an adenovirus is used as an expression vector, this coding sequence may be ligated to an adenovirus transcription / translation control complex (eg, the late promoter and three leader sequences). This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region (eg, E1, E3, or E4 region) of the viral genome results in a recombinant virus that is capable of growing and capable of expressing RNA in the infected host.

  Certain initiation signals can also be used for more efficient translation using the vRNAPs of the invention. These signals include the ATG start codon and adjacent sequences. Exogenous control signals (including the ATG start codon) may further need to be provided. One skilled in the art can easily determine this and can easily provide the required signal. It is well known that the start codon must be in reading frame and in frame (ie, in-phase) of the desired coding sequence to ensure translation of the entire insert. Control signals and initiation codons can be of various origins (both natural or synthetic) and expression efficiency can be enhanced by the inclusion of appropriate transcription enhancing elements and transcription terminators.

  In eukaryotic expression, it is also typical to incorporate an appropriate polyadenylation site (eg, 5′-AATAAA-3 ′) into the transcription unit if it is not contained within the originally cloned segment. Desired. Typically, the poly A addition site is located from about 30 nucleotides to about 2000 nucleotides “downstream” of the termination site of the proteinaceous molecule located prior to transcription termination.

  Long-term, high-yield production and stable expression of recombinant vRNAP protein, polypeptide or peptide is preferred. For example, cell lines that stably express constructs encoding vRNAP proteins, polypeptides or peptides can be engineered. Rather than using an expression vector containing a viral origin of replication, the host cell is controlled by appropriate expression control elements (eg, promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and a vector controlled by a selectable marker. Can be transformed. Following introduction of the foreign DNA, the engineered cells can be grown for 1-2 days in a concentrated medium, which is then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cell to stably integrate the plasmid into these chromosomes, which then can be cloned and expanded into a cell line. Grow to form.

A number of selection systems can be used, in herpes simplex virus thymidine kinase (tk) gene, hypoxanthine-guanine phosphoribosyltransferase (hgprt) gene and adenine phosphoribosyl in tk ⁻ cells, hgprt ⁻ cells or aprt ⁻ cells, respectively. Examples include, but are not limited to, transferase (aprt) genes. Also, antimetabolite resistance is dihydrofolate reductase (dhfr) (which confers resistance to methotrexate); gpt (which confers resistance to mycophenolic acid); neomycin (neo) (which is an aminoglycoside) Confer resistance to G-418); and hygromycin (which confers resistance to hygromycin).

  Large scale suspension culture of bacterial cells in a stirred tank is a common method for the production of recombinant proteinaceous molecules. Two suspension culture reactor designs (stirred reactor and airlift reactor) are widely used. The agitation design has been successfully used at 8000 liter capacity for the production of interferon. Cells are grown in stainless steel tanks having a height-diameter ratio of 1: 1 to 3: 1. The culture is usually mixed using one or more stirrers of blade disk type or ship propeller pattern type. A stirrer system that provides less shear than the blade is described. Agitation can be driven either directly or indirectly by a magnetic coupling drive. Indirect drive reduces the risk of microbial contamination through the seal against the stirrer shaft.

  Airlift reactors for microbial fermentation rely on a gas stream that mixes and oxygenates the culture. This gas stream enters the riser section of the reactor and drives the circulation. The gas separation at the culture surface that yields a dense liquid without gas bubbles moves downward in the downcomer compartment of the reactor. The main advantage of this design is simplicity and lack of need for mechanical mixing. Typically, this height-diameter ratio is 10: 1. Airlift reactors scale up relatively easily, have good mass transfer of gases, and produce relatively low shear forces.

  It is contemplated that a vRNAP protein, polypeptide or peptide of the invention can be “overexpressed”, ie expressed at an increased level relative to its natural expression in a cell. Such overexpression can be assessed by various methods, including radiolabeling and / or proteinaceous molecular purification. However, simple and direct methods are preferred, for example methods relating to quantitative analysis (eg densitometric scanning of the resulting gel or blot) following SDS / PAGE and proteinaceous composition staining or Western blotting. A specific increase in the level of a recombinant protein, polypeptide or peptide compared to the level in a natural cell is relatively rich in specific proteinaceous molecules relative to other proteins produced by the host cell, and For example, it is overexpressed because it is visible on the gel.

(IV. Method of gene transfer)
In order to mediate the action of the transgene in the cell, it is necessary to introduce the expression construct (eg, therapeutic construct) of the present invention into the cell. Such transfer may use viral or non-viral methods of gene transfer. This section provides a discussion of methods and compositions for gene or nucleic acid introduction, including the introduction of antisense sequences.

  The vRNAP gene is incorporated into a viral vector that mediates gene transfer into the cell. Additional expression constructs encoding EcoSSB and other therapeutic agents as described herein also provide for viral transduction using infectious viral particles, eg, by transformation with an adenoviral vector of the invention. Can be introduced. Alternatively, a retrovirus, bovine papilloma virus, adeno-associated virus (AAV), lentiviral vector, vaccinia virus, polyoma virus, or infectious virus engineered to express a specific binding ligand can be used. Similarly, non-viral methods are used, including but not limited to injection, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and direct delivery of DNA such as by microparticle guns. obtain. Thus, in one example, viral infection of cells is used to deliver therapeutically important genes to cells. Typically, the virus is simply exposed to a suitable host cell under physiological conditions that allow viral uptake.

  Microinjection can be used for delivery to cells. Microinjection involves the insertion of a substance (eg, RNA) into a cell through a microelectrode. Typical applications include injection of drugs in molecular biology research, histochemical markers (eg, horseradish peroxylase or lucifer yellow) and RNA or DNA injection. Either hydrostatic pressure (pressure injection) or current (iontophoresis) is used to exclude material that has passed through very fine electrode tips.

(V. Proteinaceous composition)
In certain embodiments, the present invention relates to a novel composition or method comprising at least one proteinaceous molecule. The proteinaceous molecule may have a sequence essentially as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 15. The proteinaceous molecule can be vRNAP or more preferably mini-vRNAP or a delivery agent. The proteinaceous molecule can also be a mutated mini-vRNAP.

  As used herein, a “proteinaceous molecule”, “proteinaceous composition”, “proteinaceous compound”, “proteinaceous chain” or “proteinaceous substance” is generally translated from a gene. A protein of greater than about 200 amino acids or a full-length endogenous sequence; a polypeptide of greater than about 100 amino acids; and / or a peptide of from about 3 to about 100 amino acids. All of the above “proteinaceous” terms may be used interchangeably herein.

  In certain embodiments, the size of the at least one proteinaceous molecule may include, but is not limited to: about 1, about 2, about 3, about 4, about 5, 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 15, about 16, about 17, about 18, About 19, about 20, about 21, about 23, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43 About 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, approx. 57 About 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82 About 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170 About 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 75, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675 About 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino molecule residues, and these Any range of amino molecule residues that can be derived from.

  As used herein, an “amino molecule” refers to any amino acid, amino acid derivative or amino acid mimetic as is known to those skilled in the art. In certain embodiments, the residues of the proteinaceous molecule are continuous without any non-amino molecules interrupting the amino molecule residue sequence. In other embodiments, the sequence can include one or more non-amino molecule moieties. In certain embodiments, the sequence of residues of a proteinaceous molecule can be interrupted by one or more non-amino molecule moieties.

  Thus, the term “proteinaceous composition” includes, but is not limited to, at least one of the 20 common amino acids in naturally synthesized proteins, or the amino acids shown in Table 5 below, at least Includes amino acid sequences that contain one modified or unusual amino acid.

特定の実施形態において、タンパク質性組成物は、少なくとも１つのタンパク質、ポリペプチドまたはペプチド（例えば、ｖＲＮＡＰまたはミニ−ｖＲＮＡＰ）を含む。さらなる実施形態において、タンパク質性組成物は、生体適合性のタンパク質、ポリペプチドまたはペプチドを含む。本明細書中で使用される場合、用語「生体適合性」とは、本明細書中に記載される方法および量に従って、所定の生物に適用されるか、または投与された場合、有意な有害な効果を生成しない物質をいう。このような有害な効果または望ましくない効果は、顕著な毒性または有害な免疫学的反応のような効果である。好ましい実施形態において、組成物を含有する生体適合性のタンパク質、ポリペプチドまたはペプチドは、一般的には、各々本質的に、毒素、病原体および有害な免疫原を含まない、哺乳動物のタンパク質もしくはペプチドまたは合成タンパク質もしくは合成ペプチドである。

In certain embodiments, the proteinaceous composition comprises at least one protein, polypeptide or peptide (eg, vRNAP or mini-vRNAP). In further embodiments, the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide. As used herein, the term “biocompatible” refers to significant adverse effects when applied to or administered to a given organism, according to the methods and amounts described herein. A substance that does not produce a positive effect. Such harmful or undesirable effects are effects such as marked toxicity or harmful immunological reactions. In preferred embodiments, the biocompatible protein, polypeptide or peptide containing composition is generally a mammalian protein or peptide, each essentially free of toxins, pathogens and harmful immunogens. Or a synthetic protein or peptide.

  Proteinaceous compositions can be made by any technique known to those skilled in the art, including expression of proteins, polypeptides or peptides through standard molecular biology techniques, from natural sources. Examples include isolation of proteinaceous compounds or chemical synthesis of proteinaceous substances. Nucleotide sequences as well as protein, polypeptide and peptide sequences for various genes have been previously disclosed and can be found in computerized databases known to those skilled in the art. One such database is the National Center for Biotechnology Information's Genbank and the GenPept database (http://www.ncbi.nlm.nih.gov/). The coding regions for these known genes can be amplified and / or expressed using techniques disclosed herein or techniques known to those skilled in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those skilled in the art.

  In certain embodiments, proteinaceous compounds can be purified. In general, “purification” refers to a specific or desired protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, These compositions then substantially retain their activity, as can be assessed by protein assays, for example, as known to those skilled in the art for the particular or desired protein, polypeptide or peptide. .

  In certain embodiments, the proteinaceous composition can comprise at least one antibody. Mini-vRNAP antibodies can include all or part of an antibody that specifically recognizes mini-vRNAP. As used herein, the term “antibody” is intended to refer to a wide range of any immunological binding agent (eg, IgG, IgM, IgA, IgD and IgE). In general, IgG and / or IgM are preferred. Because IgG and / or IgM are the most common antibodies in physiological situations, and because they are most easily made in an experimental environment.

The term “antibody” is used to refer to any antibody-like molecule having an antigen binding region, and as antibodies, antibody fragments (eg, Fab ′, Fab, F (ab ′) ₂ , single domain antibodies (DAB ), Fv, scFv (single chain Fv), etc. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Are well known in the art (see, eg, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, incorporated herein by reference).

  It is contemplated that virtually any protein, polypeptide or peptide containing component can be used in the compositions and methods disclosed herein. However, it is preferred that the proteinaceous material is biocompatible. In certain embodiments, the formation of a more viscous composition is that the high viscosity allows the composition to be applied more accurately or easily to the tissue and maintain contact with the tissue throughout the procedure, in that It is assumed to be advantageous. In such cases, the use of a peptide composition, or more preferably a polypeptide or protein composition, is contemplated. Viscosity ranges include, but are not limited to, about 40 poise to about 100 poise. In certain aspects, a viscosity of about 80 poise to about 100 poise is preferred.

  Although proteins and peptides suitable for use in the present invention can be self proteins or peptides, the present invention is clearly not limited to the use of such self proteins. As used herein, the term “self protein, polypeptide or peptide” refers to a protein, polypeptide or peptide that is derived from or obtained from an organism. Organisms that can be used include but are not limited to cattle, reptiles, amphibians, fish, rodents, birds, canines, felines, fungi, plants, or prokaryotes, and selected animals Or a human subject is preferred. The “self protein, polypeptide or peptide” can then be used as a component of a composition intended for application to a selected animal or human subject. In certain aspects, the autologous protein or peptide is prepared, for example, from the entire plasma of a selected donor. The plasma is placed in a tube and placed in a freezer at about −80 ° C. for at least about 12 hours and then centrifuged at about 12,000 × g for 15 minutes to obtain a precipitate. This precipitate (eg, fibrinogen) can be stored for up to about a year (Oz, 1990).

(VI. Protein purification)
In order to prepare compositions containing vRNAP or mini-vRNAP, it is desirable to purify these components or variants. Purification of mini-vRNAP (SEQ ID NO: 4) can be done in two steps using an affinity column. Since the mini-vRNAP of SEQ ID NO: 6 has been modified to include a His tag, purification can be done in one step when using a metal affinity column (eg, a column using nickel, cobalt or zinc). The full-length vRNAP of SEQ ID NO: 15 is also His-tagged for purification.

  In accordance with one embodiment of the present invention, purification of a peptide comprising vRNAP can ultimately be utilized to operably link to this domain using a selection agent. Protein purification techniques are well known to those skilled in the art. These techniques relate, at a certain level, to the crude fractionation of the cellular environment for polypeptide and non-polypeptide fractions. The polypeptide of interest in which the polypeptide is separated from other proteins can be further purified using chromatographic and electrophoretic techniques to achieve incomplete or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method for purifying peptides is affinity chromatography.

  Tags (eg, hexahistidine (6-His, HHHHHH), FLAG (DYKDDDDK), hemagglutinin (HA, YPYDVPDYA) and c-myc (EQKLISEEDL)) can be used for protein and peptide purification and detection. Other tags have also been created, most of which are very small and contain only a few amino acids and thus appear to have little effect on the conformation of the mature protein or peptide. These small tags do not require any special conformation to be recognized by the antibody. Systems for protein purification using these tags include the FLAG fusion system marketed by NTA resin (6-His) or IBI (FLAG), where the fusion protein is affinityd on an antibody column. Purified.

  Certain aspects of the invention relate to purification and, in certain embodiments, to substantial purification of the encoded protein or peptide (eg, vRNAP). As used herein, the term “purified protein or peptide” is intended to refer to a composition that can be isolated from other components, where the protein or peptide is obtained in nature. Purified to any degree for possible conditions. Thus, purified proteins or peptides also refer to proteins or peptides that are independent of the environment in which they can occur in nature.

  In general, “purified” refers to a protein or peptide composition (eg, vRNAP) that has been subjected to fractionation to remove various other components, and these compositions are expressed as such. Substantially retain the biological activity produced. When the term “substantially purified” is used, the nomenclature is that the protein or peptide forms the major component of the composition, eg, about 50%, about 60%, about 70% in the composition. %, About 80%, about 90%, about 95% or more of a protein.

  Various methods for quantifying the degree of protein or peptide purification are known to those of skill in the art in light of the present disclosure. These methods include, for example, determining the specific activity of the active fraction or assessing the amount of polypeptide in the fraction by SDS / PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare this activity with the specific activity of the initial extract, and then to calculate the degree of purity. It is evaluated by the number of “-fold purification” in the text. The actual units used to represent the amount of activity will, of course, depend on the particular assay technique selected after purification and whether the expressed protein or peptide exhibits detectable activity.

  Various techniques suitable for use in protein purification are well known to those skilled in the art. These techniques include, for example, centrifugation after precipitation or heat denaturation using ammonium sulfate, PEG, antibodies, etc .; chromatography steps (eg, ion exchange, gel filtration, reverse phase, hydroxyapatite and affinity chromatography); isoelectric Focusing techniques; gel electrophoresis; and combinations of these and other techniques. In general, as is known in the art, the order in which the various purification steps are performed can be altered, or certain steps can be omitted, and preparation of substantially purified proteins or peptides It is conceivable that further appropriate methods can be brought about.

  There is no general requirement that the protein or peptide always be provided in their most purified state. In fact, it is contemplated that products that are substantially less purified have utility in certain embodiments. Partial purification can be accomplished by using fewer purification steps by combining or utilizing different forms of a similar general purification scheme. For example, it is understood that cation exchange column chromatography performed using an HPLC instrument generally results in a “-fold” higher purification than similar techniques using low pressure chromatography systems. Methods that exhibit a lower degree of relative purification may have advantages in the total recovery of the protein product or in maintaining the activity of the expressed protein.

  It is known that migration of polypeptides can sometimes vary significantly in different SDS / PAGE conditions (Capaldi et al., 1977). Thus, it is understood that under different electrophoresis conditions, the apparent molecular weight of a purified or partially purified expression product can vary.

  Ion exchange chromatography is a preferred separation method. It is also preferred to use a column resin (eg, metal affinity chromatography resin TALON). TALON resin has enhanced separation power for polyhistidine tagged proteins. This results in higher purity with less effort. TALON uses cobalt (a positive metal that has a significantly higher affinity for polyhistidine-tagged proteins and a lower affinity for other proteins). Often, no recognizable binding of host proteins occurs and no separate washing steps are required. The binding properties of cobalt allow for protein elution under mild pH conditions that protect protein integrity.

  Further concentration of the protein can be done with an anion exchange column (eg, MonoQ column, high resolution, anion exchange column). This column operates at a pressure below 5 MPa, has a high capacity and gives a very high chromatographic resolution.

  High performance liquid chromatography (HPLC) is characterized by a very rapid separation with surprising peak resolution. This is achieved by the use of very fine particles and high pressure to maintain a proper flow rate. Separation can be achieved in minutes or at most in one hour. Furthermore, only a very small sample volume is required. This is because the particles are so small and close-packed that the void volume is a very small percentage of the total volume. Also, the concentration of the sample need not be so high. This is because the band is so narrow that there is very little dilution of the sample.

  Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that depends on the size of the molecule. The theory that supports gel chromatography is that columns prepared with microparticles of inert material containing pores, depending on the size of the molecule, allow larger molecules to pass through or around the pores. Separation from smaller molecules. Unless the material forming the particles adsorbs molecules, the only factor that determines the flow rate is size. Thus, molecules are eluted from the column as the size decreases as long as the shape is relatively constant. Gel chromatography is excellent for separating molecules of different sizes. This is because separation is independent of all other factors (eg, pH, ionic strength, temperature, etc.). Elution volume is related to a simple matter with respect to molecular weight, with virtually no adsorption and little zone broadening.

  Affinity chromatography, a particularly efficient method of purifying peptides, is a chromatographic procedure that relies on the specific affinity between the substance to be isolated and the molecule that can specifically bind to that substance. It is. This is a receptor-ligand type interaction. The column material is synthesized by covalently binding one of the binding partners to an insoluble matrix. Thus, the column material can specifically adsorb the material from the solution. Elution occurs by changing conditions (eg, changing pH, ionic strength, and temperature) relative to conditions where binding does not occur. Tags as described herein above can be used in affinity chromatography.

  The matrix should be a substance that itself does not adsorb molecules to any significant extent and has a wide range of chemical, physical and thermal stability. The ligand should be bound in such a way as not to affect its binding properties. The ligand should also provide relatively tight binding, and this should be able to elute the material without destroying the sample or ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. Production of antibodies suitable for use in accordance with the present invention is described below.

  The affinity column can have an N4 promoter where the vRNAP protein or mini-vRNAP protein recognizes binding to the matrix. This column is suitable for use for the purification of polymerases that do not have additional tags (eg, histidine tags).

(VII. Separation method, quantification method, and identification method)
After synthesis of RNA, it may be preferable to separate several different length amplification products from each other and from its template and excess primers.

(A. Gel electrophoresis)
In one embodiment, amplification products are separated by agarose, agarose-acrylamide, or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., 1989).

(B. Chromatographic technology)
Alternatively, chromatographic techniques can be used to effect separation. Many types of chromatography (adsorption, partitioning, ion exchange and molecular sieving) that can be used in the present invention, and many specialized techniques for using them (column chromatography, filter paper chromatography, thin-layer chromatography) (Including chromatography and gas chromatography) (Freifelder, 1982). In yet another alternative, labeled cDNA products (eg, biotin labeled or antigen labeled) can be captured with beads carrying avidin or antibody, respectively.

(C. Microfluidic technology)
Microfluidic technology includes ACLARA Biosciences Inc. Or microcapillaries designed by Caliper Technologies Inc. Separation on platforms such as LabChip ^™ “Liquid Integrated Circuits” manufactured by These microfluidic platforms require only nanoliter volumes of sample as opposed to microliter volumes required by other separation techniques. Miniaturization of some of the processes involved in genetic analysis has been accomplished using microfluidic devices. For example, published PCT application number WO 94/05414 to Northrup and White (incorporated herein by reference) reports an integrated micro PCR ^TM device for collecting and amplifying nucleic acids from specimens. To do. US Pat. No. 5,304,487 to Wilding et al. And US Pat. No. 5,296,375 to Krickka et al. Discuss devices for collection and analysis of cell-containing samples and are incorporated herein by reference. Is done. US Pat. No. 5,856,174 describes an apparatus that combines various processes and analytical operations involved in nucleic acid analysis and is incorporated herein by reference.

(D. Capillary electrophoresis)
In some embodiments, it may be desirable to provide additional or alternative means for analyzing the amplified gene. In these embodiments, it is contemplated that microcapillary arrays are used for analysis.

Microcapillary array electrophoresis generally involves the use of narrow capillaries or channels, which may or may not be filled with a specific separation medium. Electrophoresis of the sample through the capillary provides a size-based separation profile for the sample. The use of microcapillary electrophoresis in separation by nucleic acid size is reported, for example, in Woolley and Mathies, 1994. Microcapillary array electrophoresis generally provides a rapid method of size-based sequencing, PCR ^TM product analysis, and restriction fragment sizing. The high ratio of surface to volume of these capillaries allows a stronger electric field to be applied across the capillaries without substantial thermal fluctuations across the capillaries, thus allowing more rapid separation. Furthermore, when combined with confocal imaging methods, these methods provide sensitivity in the attomole range, which is comparable to the sensitivity of radial sequencing methods. Microfabrication of microfluidic devices (including microcapillary electrophoresis devices) is described, for example, in Jacobsen et al., 1994; Effenhauser et al., 1994; Harrison et al., 1993; Effenhauser et al., 1993; Manz et al., 1992; , 094,824. Typically, these methods include micron scale photolithographic etching on silica, silicon or other crystalline substrates or chips and can be readily adapted for use in the present invention. In some embodiments, the capillary array can be manufactured from the same polymeric materials as described for manufacturing the body of the device using the injection molding techniques described herein.

  Tsuda et al., 1990 describe a rectangular capillary that is an alternative to a cylindrical capillary glass tube. Some advantages of these systems are their efficient heat loss due to the large ratio of height to width (and hence the high ratio of surface to capacity), and their relative to optical detection mode on the column. High detection sensitivity. These flat separation channels have the ability to perform secondary subdivision where one force is applied across the separation channel and the sample zone is detected by use of a multi-channel array detector.

  In many capillary electrophoresis methods, capillaries (eg, fused silica capillaries or channels that are etched, machined, or shaped into a flat substrate) are filled with a suitable separation / sieving matrix. Typically, various sieve matrices are known in the art and can be used in microcapillary arrays. Examples of such a matrix include, for example, hydroxyethyl cellulose, polyacrylamide, agarose and the like. In general, the particular gel matrix, running buffer and running conditions maximize the separation characteristics of a particular application (eg, size of nucleic acid fragments, required resolution, and presence of native or native nucleic acid molecules). Selected as For example, the running buffer may contain a denaturing agent, a chaotropic agent (eg, urea, etc.) to denature the nucleic acid in the sample.

(E. Mass spectrometry)
Mass spectrometry is a means of “weighing” individual molecules by ionizing the molecules under reduced pressure and “flying” them by volatilization. Under the influence of a combination of electric and magnetic fields, these ions follow an orbit depending on their individual mass (m) and charge (z). For low molecular weight molecules, mass spectrometry is part of the conventional physico-organic chemical repertoire for the analysis and characterization of organic molecules by determining the mass of the parent molecular ion. Furthermore, by arranging collisions between this parent molecular ion and other particles (for example, argon atoms), the molecular ions are fragmented by so-called collisional dissociation (CID) to form secondary ions. Fragmentation patterns / paths very often allow the derivation of detailed structural information. Other applications of mass spectrometry known in the art are found in Methods in Enzymology, Volume 193: “Mass Spectrometry” (JA McCrossey, Ed.), 1990, Academic Press, New York. Can be done.

  Due to the obvious analytical advantages of mass spectrometry in providing high detection sensitivity, accuracy of mass measurement, detailed structural information and speed with CID combined with MS / MS configuration, and online data transmission to a computer There is considerable interest in using mass spectrometry for structural analysis of nucleic acids. For a review summarizing this area, see K. H. Schram (1990); F. Crain (1990). The biggest challenge for applying mass spectrometry to nucleic acids is the difficulty of volatilization of these highly polar biopolymers. Thus, "sequencing" is the parent molecular ion via CID in an MS / MS configuration that utilizes fast atom bombardment (FAB mass spectrometry) or plasma desorption (PD mass spectrometry), especially for ionization and volatilization. And thus limited to low molecular weight synthetic oligonucleotides by confirming already known sequences or confirming known sequences through the generation of secondary ions (fragment ions) . As an example, the application of FAB to the analysis of protected dimer blocks for chemical synthesis of oligodeoxynucleotides has been described (Koster et al., 1987).

  Two ionization / desorption techniques are electrospray / ionspray (ES) and matrix-assisted laser desorption / ionization (MALDI). ES mass spectrometry was introduced by Fenn et al., 1984; WO 90/14148, and this application has been summarized in a review article (RD Smithih et al., 1990; B. Ardrey, 1992). Quadrupoles are most frequently used as mass spectrometers. The determination of the molecular weight in a femtomolar amount sample is very accurate due to the presence of multiple ion peaks, all of which can be used for mass calculations.

  MALDI mass spectrometry, in contrast, can be particularly attractive when a time-of-flight (TOF) configuration is used as a mass spectrometer. MALDI-TOF mass spectrometry was introduced by Hillenkamp et al. (1990). Thus, in most cases, multiple molecular ion peaks do not occur using this technique, and in principle the mass spectrum looks simpler compared to ES mass spectrometry. DNA up to a molecular weight of 410,000 daltons can be desorbed and volatilized (Williams et al., 1989). More recently, the use of infrared lasers (IR) in this technology (as completely different from UV lasers) has led to larger nucleic acids (eg, synthetic DNA, restriction fragments of plasmid DNA, and sizes up to 2180 nucleotides). RNA transcripts) have been shown to provide mass spectrometry (Berkenkamp et al., 1998). Berkenkamp et al., 1998 also describes how DNA and RNA samples can be analyzed by limited sample purification using MALDI-TOR IR.

  Japanese Patent 59-131909 describes an apparatus for detecting nucleic acid fragments separated by either electrophoresis, lipid chromatography or high speed gel filtration. Mass spectrometric detection is achieved by incorporating into the nucleic acid atoms that are not normally present in DNA (eg, S, Br, I or Ag, Au, Pt, Os, Hg).

(F. Energy transfer)
Labeling hybridization oligonucleotide probes with fluorescent labels is a well-known technique in the art and is a sensitive non-radioactive method for facilitating detection of probe hybridization. More recently developed detection methods use a fluorescence energy transfer (FET) process for the detection of probe hybridization, rather than direct detection of fluorescence intensity. An FET has an absorption spectrum of one (acceptor) that overlaps the emission spectrum of the other (donor) between a donor fluorophore and an acceptor dye (which may or may not be a fluorophore). And when these two dyes are in close proximity. Dyes having these characteristics are referred to as donor / acceptor dye pairs or energy transfer dye pairs. The excited state energy of the donor fluorophore is transferred to adjacent acceptors by dipole interactions induced by resonant dipoles. This causes quenching of donor fluorescence. In some cases, if the acceptor is also a fluorophore, its fluorescence intensity can be enhanced. The efficiency of energy transfer is highly dependent on the distance between the donor and acceptor, and formulas have been developed to predict these relationships (Forster, 1948). The distance between the donor dye and the acceptor dye that is 50% energy efficient is referred to as the Forster distance (R _O ). Other mechanisms for fluorescence quenching are also known, including charge transfer and collisional quenching, for example.

  Energy transfer and other mechanisms that cause quenching depending on the interaction of two closely adjacent dyes are attractive tools for determining or identifying nucleotide sequences. This is because such assays can be performed in a homogeneous format. A homogeneous assay format is simpler than conventional probe hybridization assays, which rely on the detection of fluorescence of a single fluorophore label. This is because heterogeneous assays generally require an additional step of separating the hybridized label from the free label. Several formats for FET hybridization assays are reviewed in Nonisotopic DNA Probe Technologies (1992).

Homogeneous methods that use energy transfer or other fluorescence quenching mechanisms for detection of nucleic acid amplification have also been described. Higuchi et al. (1992) disclose a method for training DNA amplification in real time by monitoring the increased fluorescence of ethidium bromide when ethidium bromide binds to double stranded DNA. The sensitivity of this method is limited. Because ethidium bromide binding is not target specific, and background amplification products are also detected. Lee et al. (1993) discloses a real-time detection method in which a doubly labeled detection probe is cleaved during PCR ^™ in a manner specific for label amplification. This detection probe is hybridized downstream of the amplification primer so that the 5 ′ → 3 ′ exonuclease activity of Taq polymerase digests the detection probe and separates the two fluorescent dyes that form the energy transfer pair. . The fluorescence intensity increases as the probe is cleaved. WO 96/21144 discloses a continuous fluorometric assay in which enzymatically mediated nucleic acid cleavage results in increased fluorescence. Although the transfer of fluorescence energy is suggested to be used in this method, it is only in the context of methods that use a single fluorescent label (which is quenched by hybridization to the label).

  Signal primers or detection probes that hybridize to target sequences downstream of the amplification primer hybridization site have been described for use in the detection of nucleic acid amplification (US Pat. No. 5,547,861). This signal primer is extended by the polymerase in a manner similar to the extension of the amplification primer. Extension of the amplification primer replaces the extension product of the signal primer in a manner that depends on the target amplification, resulting in a double-stranded secondary amplification product that can be detected as an indicator of target amplification. The secondary amplification product generated from the signal primer contains various labels and reporter groups, restriction sites in the signal primer (which are cleaved to yield a characteristic size fragment), capture groups, and structural properties (eg, (Recognition sites for triple helix and double stranded DNA binding proteins).

  Many donor / acceptor dye pairs known in the art can be used in the present invention. These include, for example, fluorescein isothiocyanate (FITC) / tetramethylrhodamine isothiocyanate (TRITC), FITC / Texas Red (Molecular Probes), FITC / N-hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC / Eosin isothiocyanate (EITC), N-hydroxysuccinimidyl 1-pyrenesulfonate (PYS) / FITC, FITC / rhodamine X, FITC / tetramethylrhodamine (TAMRA) and the like. The selection of a particular donor / acceptor fluorophore pair is not critical, for the energy transfer quenching mechanism it is only necessary that the emission wavelength of the donor fluorophore overlaps with the absorption wavelength of the acceptor (ie , There must be sufficient spatial overlap between these two dyes to allow efficient energy transfer, charge transfer or fluorescence quenching). p- (Dimethylaminophenylazo) benzoic acid (DABCYL) effectively quenches fluorescence from adjacent fluorophores (eg, fluorescein or 5- (2′-aminoethyl) aminonaphthalene (EDANS)), non-fluorescent It is a fluorescent acceptor dye. Any dye pair that causes fluorescence quenching in the detection nucleic acid of the invention is suitable for use in the methods of the invention, regardless of the mechanism by which quenching occurs. Both end labeling and internal labeling methods are known in the art and can be routinely used to link donor and acceptor dyes at their respective sites in the detection nucleic acid.

(G. In vitro study)
The synthetic RNAs of the invention can be used for in vitro studies of spliceosome assays, splicing reactions, or antisense experiments.

  The spliceosome is a large multi-subunit complex composed of small nuclear ribonucleoprotein particles (snRNP). There are a total of 5 snRNAs: U1, U2, U4, U5, and U6, which are small and rich in uridine. Each snRNA has one or two of these RNAs. In addition to catalyzing the splicing reaction, the spliceosome retains the intermediate product, locates the splice site for precise ligation of the exons, and allows the exons to diffuse after cleavage and prior to ligation. To prevent. Spliceosome catalysis is a competitive cleavage / ligation reaction in which 2′-OH at branch site A attacks the 5 ′ splice site to form a 2′-5 ′ phosphodiester bond with the first nucleotide of the intron. including. The 3'-OH obtained at the end of the 5 'exon attacks the 3' splice site, unraveling the intron lasso form, and linking the two exons together with a normal 3'-5 'phosphodiester bond Let At least 50 different proteins are involved in spliceosome assembly and function. In group I and group II introns, splicing is improved (rate and accuracy) by protein factors (Coetze et al., 1994; Mohr et al., 1994).

(VIII. Kit)
Any composition described herein can be included in the kit. In a non-limiting example, vRNAP, or more preferably mini-vRNAP, derivatized mini-vRNAP, mutant vRNAP and / or additional agents may be included in the kit. Thus, the kit contains the mini-vRNAP, derivatized mini-vRNAP, variant vRNAP and / or additional agent of the present invention in a suitable container means. We anticipate other components that can be included in the kit. These include immunodetection agents (eg, monoclonal and polyclonal antibodies conjugated to peroxidase and alkaline phosphatase), immunoprecipitation reagents (eg, beads conjugated to protein A or protein G), immune cell proliferation reagents (eg, TALON Or monoQ column) cloning reagents for manipulating expression vectors, and protein expression reagents including, but not limited to, prokaryotic and eukaryotic cells for protein expression.

  This kit can be used to adjust a composition of the invention (standard curve for detection assay) of appropriately aliquoted vRNAP, mini-vRNAP, derivatized mini-vRNAP, mutant vRNAP and / or additional agents. As such, whether labeled or unlabeled). The components of the kit can be packaged either in aqueous media or in lyophilized form. The container means of the kit generally comprises at least one vial, test tube, flask, bottle, syringe or other container means in which the ingredients can be placed and preferably suitably aliquoted. If more than one component is present in the kit, the kit can also generally include a second, third, or other additional container, in which additional components can be placed separately. However, various combinations of components can be included in the vial. The kits of the present invention also typically comprise means for containing vRNAP, lipids, additional agents, and any other reagent containers within closed limits for commercial purposes. Such containers may include injection molded or blow molded plastic containers in which the desired vials are held.

  However, the components of this kit can be provided as a dry powder. When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is anticipated that the solvent may also be provided in another easy means.

  The kits of the present invention also typically comprise means for containing vials within closed limits, such as injection molded and / or blow molded plastic containers, for commercial purposes, wherein: The desired vial is retained.

  As used herein, “a” or “an” as used herein may mean one or more. As used herein, when used in the claims, the terms “a” or “an” when used in combination with the term “comprising” include one or more than one. Can mean. As used herein, “another” may mean at least a second or more.

(IX. Examples)
The following examples are included to demonstrate preferred embodiments of the invention. The techniques described in the following examples correspond to the techniques found by the inventors to work well in the practice of the present invention, and thus can be considered to build a preferred mode for their practice. Should be understood by those skilled in the art. However, one of ordinary skill in the art, in view of the present disclosure, may make many changes in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the invention. You should understand that.

(Example 1)
(Identification of transcriptional activation domain of N4 virion RNA polymerase)
To determine the mammalian domain possessing RNA polymerase activity, controlled proteolysis was performed followed by catalytic (transcription) self-labeling (Hartmann et al., 1988). When RNA polymerase is incubated with a benzaldehyde derivative of the starting nucleotide, the benzaldehyde group forms a Schiff base with the ε-amino group of lysine located within 12 of the nucleotide binding site. The cross-linking step was performed in the presence of the DNA template. This is because it stimulates the binding of the starting nucleotide. The unstable Schiff base was converted to a stable secondary amine by reduction under mild conditions using sodium borohydride, which was accompanied by the co-reduction of unreacted benzaldehyde derivatives. Subsequent addition of template-specific α- ³² P-labeled NTP results in the formation of phosphodiester bonds and catalytic self-labeling of transcriptionally active polypeptides. Controlled trypsin proteolysis of vRNAP was performed, followed by catalytic self-labeling and analysis with SDS-PAGE (FIG. 3A). Initially, three proteolytic fragments are produced, two of which are catalytically active. Upon further incubation with triprin, a single stable transcriptionally active product (approximately 1,100 amino acids long) remains. The N-terminal sequence of the first three proteolytic fragments (FIG. 3B) corresponds to a stable active polypeptide (mini-vRNAP) in the middle 1/3 of vRNAP (region containing the three motifs described above) (FIG. 2A, SEQ ID NOs: 3-4).

(Example 2)
(Cloning and purification of N4 mini-vRNAP)
ORFs of full-length and mini-vRNAP (SEQ ID NOs: 6 and 15) were cloned using an N-terminal hexahistidine tag under the control of pBAD (FIG. 4). The mini vRNAP domain was cloned into a pBAD B expression plasmid (purchased from Invitrogen). Five restriction enzyme sites in pBAD B were changed; the SnaI site was converted to an HpaI site and the PflMI and EcoRV sites were destroyed, all by site-directed mutagenesis. BstBI and HindIII sites were destroyed by enzymatic digestion followed by Klenow treatment and ligation. FIG. 5 (left side) shows E.I. The relative amounts of full-length and mini-vRNAP proteins purified from the E. coli BL21-derived cells on a TALON column are shown. The cloned mini vRNAP is expressed at a level 100 times higher than the cloned full length vRNAP. Further enrichment on the MonoQ column reveals that mini vRNAP is stable after incubation, as opposed to full length vRNAP (Figure 5, right). At least 10 mg of mini vRNAP at a concentration of 20 mg / ml was obtained from 1 L of induced cells in only two purification steps (TALON and MonoQ mini columns). A version of mini-vRNAP that was not tagged with histidine was also cloned (SEQ ID NO: 4). In this case, the enzyme was purified from the derived extract of induced cells in two steps (promoter DNA affinity column and MonoQ).

  Mini vRNAP has a high binding affinity (Kd = 1 nM) for N4 promoter-containing DNA oligonucleotides. This property was used for purification on a DNA affinity column of histidine-tagged mini vRNAP (SEQ ID NO: 4). Columns were prepared by adsorbing 5 'biotinylated N4 promoter-containing DNA oligonucleotides to the matrix of a 1 ml HiTrap streptavidin column (Pharmacia / Amersham catalog number 17-5112-01) according to the manufacturer's instructions. Debris-free sonication of bacterial cells expressing mini-vRNAP was passed through the column. In order to bind the mini-vRNAP to the DNA affinity column, the pH of the extraction and binding / washing buffer should be between 5-9 and the NaCl concentration should be between 50 mM and 2M. . Nucleases in the extract are inhibited by 2 mM EDTA. After washing the column, the mini vRNAP was eluted with warm water (25 ° C.); the elution temperature was raised from 4 ° C. to 25 ° C. to increase the recovery of mini vRNAP. For complete elution, the temperature can be raised to 43 ° C. without significantly changing the quality of the preparation. Elution under these conditions occurs due to metal ion removal and subsequent promoter hairpin metallization and desorption of mini-vRNAP. Different DNA oligonucleotides (SEQ ID NOs: 16-19) containing variants of the P2 promoter were used in DNA affinity columns and tested in mini vRNAP affinity purification. The best yield was achieved using the DNA oligonucleotide of SEQ ID NO: 16. However, the DNA oligonucleotides of SEQ ID NO: 19-20 require a lower temperature than the DNA of the oligonucleotide of SEQ ID NO: 16 for complete elution of the protein, which is the lower thermal stability of each promoter hairpin. Matches.

  Up to 1 mg of mini vRNAP with 90% purity is added to 100 ml of E. coli expressing mini vRNAP. E. coli culture composition extracts were obtained in a single purification step using a 1 ml DNA affinity column. The binding capacity of the DNA affinity column was not detectably reduced by multiple uses.

(Example 3)
(EcoSSB effect on transcription of single-stranded template)
We have previously shown that EcoSSB is required for N4 vRNAP transcription in vivo (Glucksmann et al., 1992). EcoSSB is unique in that it does not lose the promoter hairpin structure, unlike other SSBs that were tested for effects on vRNAP transcription (Glucksmann-Kuis et al., 1996). Recently, we reviewed the effect of EcoSSB on vRNAP transcription of single-stranded templates. FIG. 6 shows transcription in the absence and presence of EcoSSB at three different ssDNA template concentrations. The extent of EcoSSB activation is template concentration dependent, with the highest activation being at low DNA template concentrations. These results suggest that EcoSSB overcomes template limitations for the ssDNA template.

To further investigate this hypothesis, the effect of adding template or EcoSSB to the transcription reaction after incubation for 20 minutes in the absence of EcoSSB was tested. The transcription reaction mixture (5-50 μl) was 20 mM Tris-HCl (pH 7.9 at 25 ° C.), 10 mM MgCl ₂ , 50 mM NaCl, 1 mM dithiothreitol, 0.01-1 μM mini-vRNAP, 1-100 nM ssDNA. Template (30-100 nt long, synthesized by Integrated DNA Technologies), 3 mM unlabeled NTP each, 0.1 mM α- ³² P NTP (1-2 Ci / mmol, NEN), and 1-10 μM E. coli. E. coli SSB was included. Incubations were for 1-80 minutes at the indicated temperature of 37 ° C. In the presence of EcoSSB, RNA synthesis increased linearly throughout the incubation period (FIG. 7C). No increase in transcription was observed over a 10 minute incubation in the absence of EcoSSB (FIG. 7A). Addition of template at 20 minutes to reactions performed in the absence of EcoSSB resulted in a dramatic increase in RNA synthesis (FIG. 7B). Addition of EcoSSB at 20 minutes caused a slow rate of transcript recovery (FIG. 7D). These results suggest that EcoSSB converts the template from a transcriptionally inactive RNA: DNA hybrid to a transcriptionally active single stranded DNA.

  To test this hypothesis, the physical state of the DNA template and the physical state of the RNA product were analyzed by native gel electrophoresis in the presence and absence of EcoSSB. In order to have effective transcription in the absence of EcoSSB, transcription was performed at a medium DNA concentration (5 nM). At this concentration, only twice the effect of EcoSSB is observed.

The result of this experiment is shown in FIG. Use either ³² P-labeled template (right panel) or ³² P-labeled NTP (left panel) to determine the status of template (right panel) or RNA product (left panel) in the absence or presence of EcoSSB. analyzed. After transcription, the mixture was divided into three samples (control sample without additives; RNA: sample with RNaseH added to specifically degrade RNA in the DNA hybrid; and to degrade single-stranded nucleic acids. The sample was further divided into a third sample to which nuclease S1 was added. In the absence of EcoSSB, both the DNA template and the RNA product are in the form of an RNA: DNA hybrid. This is because the RNA product is sensitive to RNase H, while the band containing DNA shows altered mobility after RNase H treatment. In the presence of EcoSSB, a significant portion of the RNA product is RNaseH resistant and therefore free, but there is an RNase sensitive band corresponding to the intermediate RNA: DNA: SSB complex. Under these conditions, the DNA is in the SSB: DNA complex state. These results indicate that EcoSSB stimulates transcription through template reuse.

  In order to define the EcoSSB region essential for vRNAP transcription activation on a single-stranded template, we tested the effect of human mitochondrial SSB (HmtSSB). This HmtSSB exhibits extensive sequence and structural homology to EcoSSB. The N-terminus of EcoSSB contains a DNA binding determinant and a tetramerization determinant, while its C-terminus is involved in interactions with other replication proteins. Hmt SSB has no effect on vRNAP transcription but does not lose promoter hairpins. Interestingly, preliminary results using mutant EcoSSB and EcoSSB-Hmt SSB chimeras suggest that the C-terminal region of EcoSSB is essential for vRNAP transcriptional activation.

(Example 4)
(Characterization of mini-vRNAP transcriptional properties)
The initial properties of full length RNA polymerase and mini-vRNAP were compared at similar molar concentrations (FIG. 9A). For this comparison, a catalytic self-labeling assay and two reaction conditions were used. The first condition used a template containing + 1C, a benzaldehyde derivative of GTP and α ³² P-ATP, or the second condition used a template containing + 1T, a benzaldehyde derivative of ATP and α ³² P-GTP. . Comparison of the results in FIGS. 9B and 9C shows that mini-vRNAP exhibits initial properties similar to full-length vRNAP. Furthermore, both enzymes distinguish dATP uptake to the same extent. Mini-vRNAP does not synthesize a failure product if the first 4 nucleotides of the transcript are composed of 50% or more G or C nucleotides.

  The elongation and termination properties of both enzymes are compared in FIG. Similar short (run-off) and termination transcripts are synthesized. In addition, EcoSSB activates transcription by both enzymes to the same level. This result indicates that if there are any specific contact sites between vRNAP and EcoSSB, those sites are present in the mini-vRNAP domain.

  The sequence of the terminator signal of vRNAP present in the N4 genome includes SEQ ID NOs: 21-26. The signals of SEQ ID NOs: 21 and 22 have been tested in vitro for single stranded templates.

The mini-vRNAP transcription rate was compared to that of T7 RNA polymerase under the same conditions using the same DNA template. The template used was linearized pET11 containing the original T7 promoter and the N4 vRNAP P2 promoter introduced via cloning. The DNA template was denatured prior to performing transcription using N4 mini-vRNAP. The concentration of T7 RNAP (Promega, catalog number P2075) and mini-vRNAP were compared using SDS-PAGE. Transcription reactions consisted of 50 nM polymerase, 100 nM DNA template, 5 × transcription buffer with T7 RNAP, and 1 mM ATP, 1 mM GTP, 1 mM CTP, and 0.1 mM [ ³² P] -UTP (1 Ci / Mmol). Each reaction mixture was divided into two parts; E. coli SSB was added in half. These mixtures were incubated at 37 ° C. and aliquots were taken at different time points. Transcripts were electrophoresed on 6% sequencing gels and the amount of radiolabeled RNA was quantified by phosphoimaging. The results indicate that: (a) T7 RNAP transcription Unaffected by the presence of E. coli SSB. (B) N4 mini-vRNAP synthesized 1.5-5 times more RNA than T7 RNAP in the presence of EcoSSB at different incubation time points.

  The optimum temperature for mini-vRNAP transcription is 37 ° C. Mini-vRNAP exhibits 70% activity at 30 ° C, 65% activity at 45 ° C, and only 20% activity at 50 ° C.

Average error frequency was evaluated. This evaluation was based on measuring the frequency of misincorporation of each of the four [ ³² P] -αNTPs into the RNA product using template ssDNA lacking the corresponding template nucleotide in the transcribed region. The following values were obtained: “No C” (SEQ ID NO: 10) ssDNA template and “No A” (SEQ ID NO: 11) for G and U misincorporation using ssDNA templates, respectively, 1/5 × 10 ⁴ ; 1/4 × 10 ⁴ for misincorporation of C using the “No G” (SEQ ID NO: 12) template; and 1/2 × for misincorporation of A using the “No T” (SEQ ID NO: 13) template. 10 ⁴ . As a comparison, the average error frequency for T7 RNAP is 1/2 × 10 ⁴ (Huang et al., 2000). Using the mispairing detection method described by Huang et al. (2000), no mis-uptake by mini-vRNAP was detected.

  The ability of mini-vRNAP to incorporate derivatized nucleotides was measured. Transcription by mini-vRNAP in the presence of 0.1-1 mM digoxigenin-11-UTP (Catalog No. 1209256, Roche), Biotin-16-UTP (Catalog No. 1388908, Roche) or non-derivatized UTP is used as a transcription template. A “control” ssDNA (SEQ ID NO: 9) was used to generate an equivalent amount of RNA product. RNA products synthesized in the presence of derivatized UTP have a higher molecular weight than RNA products synthesized in the presence of non-derivatized UTP, and the difference corresponds to the difference in mass of UTP used. Several derivatives are tested (ie 2'-fluoro-ribonucleoside triphosphates, dideoxynucleoside triphosphates). A fluorescent analog, fluorescein-12-UTP (Roche catalog number 1427857), a template encoding a 51 nucleotide transcript containing a series of U and a nucleotide mixture containing only ATP, CTP, GTP and fluorescein-12-UTP. Used and tested. Transcription was only 3% of that achieved with UTP, biotin-6-UTP or digoxigenin-11-UTP under the same reaction conditions. However, higher yields of fluorescent analog incorporation are expected to occur in the presence of non-derivatized UTP or on templates with other sequence compositions.

(Example 5)
(Sequencing determinant of mini-vRNAP promoter binding)
The three N4 early promoters present in the N4 genome contain a pair of Cs separated by 4 nucleotides from the base of the 5 bp promoter stem. In the preferred promoter P2, these 4 bases are A and C is followed by T. Preferably, mini-vRNAP uses a 17 nucleotide promoter sequence located immediately upstream of the transcription start site. The promoter for N4 vRNA polymerase is described in Haynes et al. (1985) and Dai et al. (1998), incorporated herein by reference. The vRNAP promoter recognition sequence and vRNAP promoter activity require a specific sequence and a hairpin structure on the template strand. The vRNAP promoters of SEQ ID NOs: 27-29 have a hairpin structure composed of a 5-7 bp stem and a 3b purine-containing loop (shown in bold in Table 6) (inverted repeats are underlined in Table 6). . The −11 position corresponds to the center of the loop, and +1 indicates the transcription start site.

本発明の他の可能なｖＲＮＡＰプロモーターは、２〜７ｂｐ長ステムと、中心および／または中心位置の隣にプリンを有する３〜５ｂループとを含む、ヘアピンを形成する任意の逆方向反復の組を含む。

Other possible vRNAP promoters of the invention include any inverted repeat set that forms a hairpin, including a 2-7 bp long stem and a 3-5b loop with purines next to the center and / or center position. Including.

To study the promoter determinant sequencing determinants, 20 base promoter oligonucleotides (including the wild type vRNAP promoter P2 sequence, substituted at all positions with one 5-iodo-dU) were used. Whenever a substitution was made in the stem, the corresponding paired base changed to A. These oligonucleotides were ³² P end labeled. It was then used to measure enzyme affinity for promoter DNA by a filter binding assay and to measure the ability to cross-link to mini-vRNAP upon UV irradiation at 320 nm. A 20 base oligonucleotide containing the wild type promoter P2 sequence binds with 1 nM Kd. Most oligonucleotides were shown to be close to wild type affinity except for oligonucleotides substituted at positions -11 (center of loop) and -8. This indicates that these positions are essential for promoter recognition (FIG. 11). Surprisingly, UV cross-linking was most effective at position -11, despite low binding affinity. This indicates specific contact with mini-vRNAP at this position. Cross-linking was also observed at the +1, +2, and +3 positions. This indicates non-specific contact with this region of the template. This is because oligonucleotides with 5-iodo-dU substitution at these positions showed wild-type binding affinity.

  The effect of changes in hairpin stem length on the ability of mini-vRNAP to bind to P2 promoter DNA was analyzed. As shown above, the wild type promoter P2 with a 5 bp stem has a Kd of 1 nM (FIG. 12, top). This stem was shortened by removal of the 3'base as shown in FIG. 12 (left). The stem can be shortened by 2 base pairs without changing the binding affinity. If two or one loop proximal base pairs remain, the binding affinity of the template is still quite present (2-10 nM). This result, surprisingly, is not unexpected as it has been shown that oligonucleotide 3′d (CGAGGGCG) 5 ′ forms an unusually stable minihairpin (Yoshizawa et al., 1997). . If one more nucleotide is removed and cannot form a loop, no binding is observed. These results indicate that loop formation is essential for vRNAP promoter recognition.

  The effect of extending the stem by the addition of a 3 'base is shown in Fig. 12 (right). The stem can be extended by 2 base pairs without changing the binding affinity. On the other hand, base pairing at the -2 position reduces binding affinity by 2 orders of magnitude, and a further reduction of 1 order of magnitude occurs due to base pairing at the -1 and +1 positions. These results indicate that the single-stranded template at positions -2, -1 and +1 is required for efficient template binding.

  All three N4 early promoters present in the N4 genome comprise a C pair separated by 4 nucleotides from the base of the 5 bp promoter stem. In promoter P2, these four bases are A and C is followed by T. In order to identify the determinants of the transcription start site, a series of templates were constructed with a single C located at different distances from position 11 of the hairpin by the addition or deletion of an A extension present in promoter P2 (FIG. 13). The affinity of these promoters for mini-vRNAP was measured by filter binding and transcription initiation was measured by catalytic self-labeling of mini-vRNAP. All templates showed similar binding affinity. However, only the template with C located 12 bases downstream from the center of the hairpin could support the initiation of transcription. This result indicates that mini-vRNAP uses this position as the transcription start site (+1).

(Example 6)
(Identification of sequence motifs essential for mini-vRNAP activity)
As shown in FIG. 2A, vRNAP includes the sequence Rx ₃ Kx _6-7 YG (referred to as motif B in Pol I and Polα DNA polymerases and T7-like RNA polymerase). To determine the relevance of this motif to vRNAP activity, two mutants (K670A and Y678F (SEQ ID NO: 8) (position number in mini-vRNAP)) were constructed by site-directed mutagenesis of mini-vRNAP. In T7-like RNA polymerase, lysine is involved in nucleotide binding and tyrosine is involved in distinguishing deoxynucleoside triphosphates, so these two positions were chosen (Maksimova et al., 1991; Bonner et al., 1992; Osumi- Davis et al., 1992). The His-tagged Y678F mini vRNAP gene (SEQ ID NO: 7) differs from the mini vRNAP domain sequence (SEQ ID NO: 3) at two positions: nucleotide 2033 (A) is replaced with T, and nucleotide 2034 (T) is Replaced with C.

  These RNA polymerase mutants were cloned under pBAD control, purified, and tested for binding ability to wild-type polymerase. Both mutant polymerases bound to promoter DNA with wild type affinity and were cross-linked to P2-DNA templates substituted with 5-iodo-dU at positions -11 and +3 with wild type affinity (FIG. 14). This indicates that these mutations do not affect promoter binding.

  Mutant enzymes were tested for their ability to support run-off transcription. The wild type enzyme and the Y678F enzyme (SEQ ID NO: 8) showed similar activity under both template excess and template restriction conditions, whereas the K670A enzyme showed reduced activity under both conditions (FIG. 15). . Under restriction template conditions, all three enzymes were activated with Eco SSB (right panel). However, the Y678F enzyme showed a reduced distinction between ribonucleoside triphosphates and deoxyribonucleoside triphosphates.

  The starting properties of the three enzymes were compared using catalytic self-labeling (FIG. 16). The K670A enzyme shows significantly reduced activity with GTP derivatives. In contrast to the wild type polymerase, the Y678F enzyme incorporates dATP with a similar efficiency to rATP in a single phosphodiester bond formation.

  Thus, the behavior of the K670A and Y678F mutant enzymes indicates that motif B is involved in the catalyst, lysine is probably required for NTP binding, and tyrosine is responsible for dNTP differentiation. These results suggest that vRNAP is a class II T7-like RNA polymerase despite extensive sequence similarity deficiencies. Recent experimental results have revealed the location of the two carboxylates (aspartic acid) involved in the catalyst.

(Example 7)
(Development of in vivo systems for in vivo expression of RNA and proteins using the mini-vRNAP and N4 vRNAP promoters)
A plasmid template was constructed using a reporter gene (β-galactosidase α-peptide) cloned under the control of the vRNAP promoter P2 present in either of two directions (FIG. 17B). The reporter construct was produced by cloning the cassette into plasmid pACYC177, which was obtained from New England Biolabs. This cassette contains a sequence (lacZ ′) encoding an approximately 30 bp long fragment starting from pT7Ac (purchased from United States Biochemical), the N4 promoter and the α fragment of lacZ. The N4 promoter and lacZ ′ were produced by oligonucleotide annealing and PCR ^TM amplification, respectively. This cassette was replaced with the pACY177 sequence located between the restriction enzyme ApaLI and BamHI cleavage sites. These reporter plasmids and recombinant full-length vRNAP or mini-vRNAP expressing plasmids are E. coli. E. coli DH5α (ΔM15) (this strain encodes β-galactosidase ω-peptide). Expression of the reporter gene (α-peptide) in this strain results in the synthesis of active β-galactosidase and the resulting production of blue colonies on X-gal plates. Transcription of α-peptide by full-length vRNAP and miniRNAP was assayed on induced X-gal medium and is shown in FIG. 17A. Introduction of full length polymerase results in small colonies that do not have β-galactosidase activity. This is not surprising since full-length vRNAP was degraded in these cells (FIG. 17C). In contrast, introduction of mini-vRNAP led to β-galactosidase activity derived only from plasmids containing detectable levels of protein (FIG. 17C) and promoter P2 in the proper orientation (FIG. 17A). These results indicate that this system is suitable for in vivo expression of RNA and proteins under the control of the mini-N4 vRNAP promoter.

  All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. The compositions and methods of the present invention have been described with reference to preferred embodiments, and changes may be made to the methods and process steps or process flows described herein without departing from the concept, spirit and scope of the present invention. It will be apparent to those skilled in the art that it can be applied. More particularly, it is clear that certain reagents, both chemical and physiologically relevant reagents, can be substituted for the reagents described herein while achieving the same or similar results. It is. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

(References)
The following references are specifically incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement the procedures or details set forth herein.

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 shows the bacteriophage N4 vRNAP promoter on a single stranded template. These promoters are characterized by conserved sequences and a 5 bp stem, 3 base loop hairpin structure. FIG. 2 shows the production of N4 vRNAP and mini vRNAP. FIG. 2A shows the following three motifs: DNA-dependent polymerase, found T / DxxGR motif (P loop), ATP / GTP binding motif present in several nucleotide binding proteins, and motif B (Rx ₃ Kx _{6 to 7-YG),} it shows a schematic view of N4 VRNAP proteins with, one of the three motifs, common to Pol I DNA polymerase and Pol alpha DNA polymerase and T7-like RNA polymerase. FIG. 2B shows mini-vRNAP. FIG. 3 shows the identification of the minimal transcriptional activation domain of N4 vRNAP by proteolytic cleavage. SDS-PAGE analysis of the products of vRNAP digestion with trypsin (FIG. 3A). N-terminal sequencing of the three early proteolytic fragments indicated that the stable active polypeptide (mini-vRNAP) corresponds to the central 1/3 of the vRNAP and that this region contains the three motifs described in FIG. 2A. Shown (FIG. 3B). FIG. 4 shows that the full-length polymerase ORF, mini-vRNAP and its mutants were cloned under pBAD control using an N-terminal hexahistidine tag. FIG. 5 shows the purification of cloned vRNAP and mini-vRNAP. The left hand side shows the relative amounts of full-size vRNAP protein and mini-vRNAP protein purified from a TALON column from an equal volume of derivatized cells. Further enrichment with a monoQ column reveals that mini vRNAP is stable after induction (right) compared to full size vRNAP. FIG. 6 shows activation of N4 vRNAP transcription by EcoSSB at three different ssDNA concentrations. The extent of EcoSSB activation was template concentration dependent, with the highest activity at low DNA template concentrations. FIG. 7 shows the effect of EcoSSB on ssDNA template regeneration. In the absence of EcoSSB, no increase in transcription was observed over 10 min incubation (FIG. 7A). Addition of the template to the reaction at 20 minutes performed in the absence of EcoSSB resulted in a dramatic increase in RNA synthesis (Figure 7B). RNA synthesis increased linearly over the incubation period (FIG. 7C). Addition of EcoSSB at 20 minutes led to a slow rate of transcription recovery (FIG. 7D). FIG. 8 shows the effect of EcoSSB on the status of template DNA and product RNA in vRNAP transcription. Native gel electrophoresis was performed in the absence and presence of EcoSSB. Transcription is performed at an intermediate (5 μM) DNA concentration, and only twice the effect of EcoSSB is observed. Either ³² P-labeled template (right panel) or labeled NTP (left panel) analyzes the status of template (right panel) or RNA product (left panel) in the absence or presence of EcoSBB Used to do. FIG. 9 shows transcription initiation by vRNAP and mini-vRNAP. Whether the starting properties of full-length and mini-vRNA polymerases were determined using a catalytic autolabeling assay and two reaction conditions (+ 1C, a benzaldehyde derivative of GTP, and a template containing α ³² P-ATP , Or + 1T, with a benzaldehyde derivative of ATP, and a template containing α ³² P-GTP) (FIG. 9A). Comparison of the results in FIG. 9B and FIG. 9C demonstrated that mini-vRNAP exhibits similar starting properties to full-size vRNAP. FIG. 10 shows the effect of EcoSSB on transcription of vRNAP and mini-vRNAP. The extension and termination properties of vRNAP and mini-vRNAP are compared. FIG. 11 shows the determination of mini vRNAP promoter contact. A 20 base oligonucleotide containing the wild type promoter P2 sequence binds with 1 nM Kd (FIG. 11A). Shows that most oligonucleotides substituted with 5-iodo-dU at specific positions are close to wild-type affinity except for oligonucleotides substituted at positions -11 (loop center) and -8. This indicates that these positions are essential for promoter recognition (FIG. 11B). UV cross-linking indicates that mini-vRNAP initially contacts at position -11. FIG. 12 shows the binding affinity of stem length promoter variants. The wild type promoter P2 with a 5 bp stem has a Kd of 1 nM (top). This stem is shortened by removal of the 3 ′ base (left). This stem can be shortened by two sets of base pairs without changing the binding affinity. The effect of lengthening the stem by addition of 3 ′ base is shown (right). This stem can be lengthened by two sets of base pairs without changing the binding affinity. FIG. 13 shows the identification of the translation start site by automatic catalyst labeling. A series of templates were constructed with a single C placed at different positions from the center of the hairpin (position -11) by addition or deletion of the As region present in promoter P2 (Figure 13A). The affinity of mini-vRNAP for these promoters was measured by filter binding and transcription initiation was measured by catalytic autolabeling of mini-vRNAP. All templates showed similar binding affinity. However, only the template with C located 12 bases downstream from the center of the hairpin could support the initiation of transcription (FIG. 13B). FIG. 14 shows UV cross-linking of mutant mini-vRNAPase to promoter oligonucleotide. Two mutants (K670A and Y678F) were tested for their ability to bind to the wild type promoter. Both mutant RNA polymerase bound to promoter DNA and mutant RNA polymerase cross-linked at positions -11 and +3 to 5-iodo-dU substituted P2 DNA template with wild type affinity and wild type The enzyme shows that these polymerase mutations do not affect promoter binding. FIG. 15 shows run-off transcription by mutant mini-vRNAPase. The wild type enzyme and Y678F (SEQ ID NO: 8) showed similar activity under both template-over and template-restricted conditions, whereas the K670A enzyme showed reduced activity under both conditions. Under restriction template conditions, all three enzymes were activated by EcoSSB (right panel). However, the Y678F enzyme showed a reduced separation between ribonucleoside triphosphate and deoxyribonucleoside triphosphate incorporation. FIG. 16 shows mutant mini-vRNAPase at the start of transcription. The starting properties of the three enzymes were compared using catalytic self-labeling. The K670A enzyme shows significantly reduced activity using GTP derivatives. The Y678F enzyme incorporates dATP as efficiently as rATP in a single phosphodiester bond formation compared to wild type polymerase. FIG. 17 shows the detection of N4 vRNAP and mini-vRNAP activity in vivo. Transcription of β-galactosidase α-peptide by full size and mini vRNAP was assayed on induced Xgal medium (FIG. 17A). A plasmid (pACYC) template was constructed with a reporter gene (α peptide of β-galactosidase) under the control of the vRNAP promoter P2 cloned in either of the two directions (FIG. 17B), while the full-length vRNAP was degraded. On the other hand, induction of mini-vRNAP led to production and accumulation of detectable levels of protein (FIG. 17C).

Claims (97)

A nucleic acid isolated, SEQ ID NO: 4, SEQ ID NO: 6, or consist region encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 8, a nucleic acid. The nucleic acid according to claim 1, wherein the nucleic acid consists of the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. 2. The nucleic acid of claim 1, wherein the nucleic acid is operably linked to a promoter. The nucleic acid according to claim 3, wherein the promoter is the N4 vRNAP promoter represented by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. The nucleic acid according to claim 3, wherein the promoter is a P2 sequence represented by SEQ ID NO: 16 or SEQ ID NO: 28. A recombinant host cell comprising a DNA segment encoding an N4 virion RNA polymerase having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6. The recombinant host cell according to claim 6, wherein the DNA segment is a single-stranded DNA segment. The recombinant host cell according to claim 6, wherein the DNA segment is a double-stranded DNA segment. The recombinant host cell according to claim 6, wherein the DNA segment encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 4. The recombinant host cell according to claim 6, wherein the DNA segment encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 6. The cells are E. coli. The recombinant host cell according to claim 6, which is an E. coli cell. A recombinant vector, comprising a DNA segment encoding an N4 virion RNA polymerase polypeptide having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6 under the control of a promoter. An isolated polynucleotide, consisting of SEQ ID NO: 3 identical or complementary sequences, polynucleotide. 2. The nucleic acid of claim 1, wherein the polypeptide has at least one histidine tag. The nucleic acid according to claim 1, wherein the polypeptide has a mutation at position Y679 of SEQ ID NO: 4 or a mutation at Y715 of SEQ ID NO: 6. A method for producing RNA comprising:
(A) obtaining N4 virion RNA polymerase;
(B) obtaining DNA;
(C) mixing the RNA polymerase and the DNA; and (d) culturing the RNA polymerase and the DNA under conditions effective to allow RNA synthesis, ,
Here, the RNA polymerase is selected from the group consisting of an RNA polymerase having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6 and a variant of the RNA polymerase having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6. Wherein the variant has a mutation at position number Y679 of SEQ ID NO: 4 or has a mutation at position number Y715 of SEQ ID NO: 6. 17. The method of claim 16, further comprising synthesizing a polynucleotide comprising a modified ribonucleotide or a modified deoxyribonucleotide. The method according to claim 16, wherein the DNA is single-stranded DNA. The method according to claim 16, wherein the DNA is a double-stranded DNA. The method of claim 16, wherein the mixing step occurs in a host cell. The host cell is E. coli. 21. The method of claim 20, wherein the method is an E. coli host cell. The method of claim 16, wherein the RNA polymerase has the amino acid sequence shown in SEQ ID NO: 4. The method according to claim 16, wherein the RNA polymerase is an RNA polymerase variant having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6.
Here, the mutant has a mutation at position number Y679 of SEQ ID NO: 4 or a mutation at position number Y715 of SEQ ID NO: 6. 24. The method of claim 23, wherein the variant is histidine tagged. The method of claim 16, wherein the RNA comprises derivatized nucleotides. The method of claim 16, further comprising using a promoter. 27. The method of claim 26, wherein the promoter is the N4 vRNAP promoter shown in SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. 28. The method of claim 27, wherein the promoter is a P2 sequence shown in SEQ ID NO: 16 or SEQ ID NO: 28. A reverse direction in which the promoter forms a hairpin having a stem of 2-7 base pairs in length and a loop of 3-5 bases and having a purine at the central position of the loop and / or next to the central position. 27. The method of claim 26, comprising a set of iterations. 27. The method of claim 26, wherein the promoter sequence is upstream of the transcription start site. The process according to claim 16, wherein step (c) is carried out at a pH between 6 and 9. 32. The method of claim 31, wherein step (c) is performed at a pH between 7.5 and 8.5. The method of claim 16, further comprising mixing Mg ⁺² or Mn ⁺² . 34. The method of claim 33, comprising mixing Mg ^{+ 2} . The method of claim 16, further defined as being performed at a temperature between 25 ° C. and 50 ° C. 36. The method of claim 35, further defined as being performed at a temperature of 30C to 45C. 37. The method of claim 36, further defined as being performed at a temperature of 32 [deg.] C to 42 [deg.] C. The method of claim 16, further comprising a step of translation. The method of claim 16, further comprising using a reporter gene. 40. The method of claim 39, wherein the reporter gene is [beta] -galactosidase [alpha] -peptide. The method of claim 16, wherein the mixing step occurs in vivo. The method of claim 16, wherein the mixing step occurs in vitro. E. further comprising the step of mixing said DNA and said polymerase with E. coli single chain binding protein (EcoSSB), an SSB protein homologous to EcoSSB or another naturally occurring or chimeric SSB protein homologous to EcoSSB. Item 17. The method according to Item 16. 44. The method of claim 43, further comprising translating the RNA. The method according to claim 16, wherein the DNA is a single-stranded linear DNA. The method according to claim 16, wherein the DNA is a single-stranded circular DNA. 47. The method of claim 46, wherein the circular DNA is bacteriophage M13 DNA. The method according to claim 16, wherein the DNA is denatured DNA. 49. The method of claim 48, wherein the denatured DNA is single stranded DNA. The method according to claim 16, wherein the RNA is purified RNA. The method of claim 16, wherein the RNA comprises a modified nucleotide. 17. A method according to claim 16, wherein mixed RNA-DNA oligonucleotides are made. 17. The method of claim 16, wherein EcoSSB is not mixed with the RNA polymerase and the DNA, and the RNA is in the form of a DNA / RNA hybrid. The method of claim 16, wherein the RNA comprises a detectable label. 55. The method of claim 54, wherein the detectable label is a fluorescent tag. 55. The method of claim 54, wherein the detectable label is biotin. 55. The method of claim 54, wherein the detectable label is digoxigenin. 55. The method of claim 54, wherein the detectable label is 2'-fluoro nucleoside triphosphate. 55. The method of claim 54, wherein the detectable label is a radioactive label. 60. The method of claim 59, wherein the radioactive label is a ³⁵ S label or a ³² P label. 55. The method of claim 54, wherein the RNA is adapted for use as a probe for blotting experiments or in situ hybridization. 17. The method of claim 16, wherein nucleoside triphosphate (NTP) is incorporated into the RNA. 64. The method of claim 62, wherein the NTP comprises a detectable label. 64. The method of claim 63, wherein the NTP is a derivatized NTP. 17. The method of claim 16, wherein deoxynucleoside triphosphate is incorporated into the RNA. 17. The method of claim 16, wherein the RNA is compatible with NMR structure determination. 68. The method of claim 66, wherein the RNA has between 10 and 1000 bases. 68. The method of claim 67, wherein the RNA has between 10 and 300 bases. 17. The method of claim 16, wherein the RNA is compatible with spliceosome assembly. 17. The method of claim 16, wherein the RNA is compatible with a splicing reaction. 17. The method of claim 16, wherein the RNA is adapted for use in antisense experiments. 17. The method of claim 16, wherein the RNA is adapted for use in probing complementary nucleotide sequences. 17. The method of claim 16, wherein the RNA is adapted for use as a probe in RNase protection studies. 17. The method of claim 16, further comprising delivering the RNA to a cell. 75. The method of claim 74, wherein the delivering step is by microinjection. 17. The method of claim 16, further comprising amplifying the RNA. A method for producing RNA comprising:
(A) obtaining N4 virion RNA polymerase;
(B) obtaining a single-stranded DNA oligonucleotide, wherein the oligonucleotide comprises an N4 virion RNA polymerase promoter sequence;
(C) mixing the RNA polymerase and the oligonucleotide; and (d) culturing the RNA polymerase and the oligonucleotide under conditions effective to allow RNA synthesis. There,
Here, the RNA polymerase has the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. 78. The method of claim 77, wherein the DNA has between 20 and 200 bases. A method for producing RNA comprising:
(A) obtaining N4 virion RNA polymerase;
(B) obtaining single-stranded DNA, wherein the DNA comprises an N4 virion RNA polymerase promoter sequence;
(C) obtaining ribonucleoside triphosphate (XTP) or derivatized ribonucleoside triphosphate;
(D) mixing the RNA polymerase, the DNA and the XTP; and (e) culturing the RNA polymerase and the oligonucleotide under conditions effective to allow RNA synthesis. Wherein the RNA is a derivatized RNA comprising a step comprising:
Here, the RNA polymerase is selected from the group consisting of an RNA polymerase having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8 and an RNA polymerase variant having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6. Wherein the variant has a mutation at position number Y679 of SEQ ID NO: 4 or has a mutation at position number Y715 of SEQ ID NO: 6. 80. The method of claim 79, wherein the RNA polymerase has the amino acid sequence shown in SEQ ID NO: 4. The RNA polymerase is a mutant of RNA polymerase having an amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 6, wherein the mutant has a mutation at position number Y679 of SEQ ID NO: 4. The method according to claim 79, or a mutation at position number Y715 of SEQ ID NO: 6. 80. The method of claim 79, wherein the RNA polymerase has an amino acid sequence set forth in SEQ ID NO: 8. A method for protein synthesis in vivo, comprising:
(A) obtaining an RNA polymerase having the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof, wherein the variant has a mutation at position No. Y679 of SEQ ID NO: 4;
(B) obtaining DNA, wherein the DNA comprises an N4 virion RNA polymerase promoter sequence;
(C) mixing the RNA polymerase and the DNA;
(D) culturing the RNA polymerase and the DNA under conditions effective to allow RNA synthesis; and (e) culturing the RNA in vivo under conditions effective to allow protein synthesis. A method comprising the steps of: 84. The method of claim 83, wherein step (e) comprises using a two plasmid system. 84. The method of claim 83, wherein step (e) comprises using a single plasmid system. 86. The method of claim 85, wherein the reporter gene and the RNA polymerase are present on the same plasmid. A method of making a mini-vRNAP comprising:
(A) a step of expressing vRNAP, wherein the vRNAP has the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or a variant thereof, wherein The variant has a mutation at position number Y679 of SEQ ID NO: 4 or has a mutation at position number Y715 of SEQ ID NO: 6; and (b) purifying the vRNAP. Claim wherein said expressing occurs in a bacterial host cell, yeast host cell, CHO host cell, Cos host cell, HeLa host cell, NIH3T3 host cell, Jurkat host cell, 293 host cell, Saos host cell, or PC12 host cell. 90. The method according to item 87. 90. The method of claim 87, further comprising using a promoter appropriate for expression in the host cell line used. 90. The method of claim 89, wherein the promoter is pBAD. 90. The method of claim 89, wherein the promoter is a promoter recognized by T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase. 90. The method of claim 89, wherein the promoter is a promoter that is recognized by an RNA polymerase from a T7-like bacteriophage. 90. The method of claim 87, wherein the vRNAP has a specific recombination sequence for use in purification. 94. The method of claim 92, wherein the vRNAP has at least one histidine tag, FLAG tag, hemagglutinin tag, or c-myc tag. 94. The method of claim 92, wherein the vRNAP has at least one histidine tag. 94. The method of claim 93, wherein the purifying step is performed in one step. 88. The method of claim 87, wherein the vRNAP has no tag.

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